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1
Loehr, B. I., R. Pontarollo, R. Rankin, L. Latimer, P. Willson, L. A. Babiuk, and S. van Drunen Littel-van den Hurk. "Priming by DNA immunization augments T-cell responses induced by modified live bovine herpesvirus vaccine." Journal of General Virology 82, no. 12 (December 1, 2001): 3035–43. http://dx.doi.org/10.1099/0022-1317-82-12-3035.
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DNA vaccines have several advantages over conventional vaccines. One of the most important characteristics is the presentation of antigen via both MHC class I and class II receptors. Although this generally results in strong T-cell responses, antibody production and protection achieved by DNA immunization are unfortunately not always adequate. In contrast, modified live virus (MLV) vaccines usually induce adequate antibody and moderate cellular responses, whereas killed vaccines tend to elicit weak immune responses in general. A DNA prime–MLV boost regimen should result in enhanced cellular immunity and possibly improved antibody production. To test this hypothesis, plasmids encoding bovine herpesvirus-1 (BHV-1) glycoproteins B and D were delivered by gene gun to the genital mucosa of cattle prior to immunization with modified live BHV-1 vaccine. The immune responses induced were compared to those of an MLV-vaccinated group and a negative control group. Although significantly enhanced T-cell responses were induced by priming with the DNA vaccine, there was no increase in antibody titres. Similar levels of protection were induced by the MLV vaccine alone and the DNA prime and MLV boost regimen, which suggests that there is no correlation between the induction of T-cell responses and protection from BHV-1 challenge.
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Mirow, Manuela, Lea Isabell Schwarze, Boris Fehse, and Kristoffer Riecken. "Efficient Pseudotyping of Different Retroviral Vectors Using a Novel, Codon-Optimized Gene for Chimeric GALV Envelope." Viruses 13, no. 8 (July 27, 2021): 1471. http://dx.doi.org/10.3390/v13081471.
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The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 106 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.
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Higuchi-Takeuchi, Mieko, Takaaki Miyamoto, Choon Pin Foong, Mami Goto, Kumiko Morisaki, and Keiji Numata. "Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria." International Journal of Molecular Sciences 21, no. 22 (November 16, 2020): 8625. http://dx.doi.org/10.3390/ijms21228625.
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Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.
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Lee, Dongwoo, Jida Liu, Hyun Jung Junn, Eun-Joo Lee, Kyu-Shik Jeong, and Dai-Wu Seol. "No more helper adenovirus: production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants." Experimental & Molecular Medicine 51, no. 10 (October 2019): 1–18. http://dx.doi.org/10.1038/s12276-019-0334-z.
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Abstract Gene therapy is emerging as an effective treatment option for various inherited genetic diseases. Gutless adenovirus (GLAd), also known as helper-dependent adenovirus (HDAd), has many notable characteristics as a gene delivery vector for this particular type of gene therapy, including broad tropism, high infectivity, a large transgene cargo capacity, and an absence of integration into the host genome. Additionally, GLAd ensures long-term transgene expression in host organisms owing to its minimal immunogenicity, since it was constructed following the deletion of all the genes from an adenovirus. However, the clinical use of GLAd for the treatment of inherited genetic diseases has been hampered by unavoidable contamination of the highly immunogenic adenovirus used as a helper for GLAd production. Here, we report the production of GLAd in the absence of a helper adenovirus, which was achieved with a helper plasmid instead. Utilizing this helper plasmid, we successfully produced large quantities of recombinant GLAd. Importantly, our helper plasmid-based system exclusively produced recombinant GLAd with no generation of helper plasmid-originating adenovirus and replication-competent adenovirus (RCA). The recombinant GLAd that was produced efficiently delivered transgenes regardless of their size and exhibited therapeutic potential for Huntington’s disease (HD) and Duchenne muscular dystrophy (DMD). Our data indicate that our helper plasmid-based GLAd production system could become a new platform for GLAd-based gene therapy.
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Niño-Sánchez, Jonatan, Li-Hung Chen, Jorge Teodoro De Souza, Sandra Mosquera, and Ioannis Stergiopoulos. "Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria." Journal of Fungi 7, no. 2 (February 9, 2021): 125. http://dx.doi.org/10.3390/jof7020125.
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Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.
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Verch, Thorsten, Zhen-Kun Pan, and Yvonne Paterson. "Listeria monocytogenes-Based Antibiotic Resistance Gene-Free Antigen Delivery System Applicable to Other Bacterial Vectors and DNA Vaccines." Infection and Immunity 72, no. 11 (November 2004): 6418–25. http://dx.doi.org/10.1128/iai.72.11.6418-6425.2004.
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ABSTRACT Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels. In vaccine development, with live vaccine vectors, this allows greater flexibility and the ability to induce larger antigen amounts through multiple gene copies. However, plasmid retention often requires antibiotic resistance markers, the presence of which has been discouraged in clinical applications by the Food and Drug Administration. Therefore, we developed a Listeria monocytogenes-Escherichia coli shuttle plasmid that is retained by complementation of d-alanine racemase-deficient mutant strains both in vitro and in vivo. Our technology potentially allows the production of antibiotic resistance marker-free DNA vaccines as well as bacterial vaccine vectors devoid of engineered antibiotic resistances. As a proof of concept, we applied the d-alanine racemase complementation system to our Listeria cancer vaccine platform. With a transplantable tumor model, we compared the efficacy of the new Listeria vector to that of an established vector containing a conventional plasmid carrying a tumor-specific antigen. Both vaccine vector systems resulted in long-term regression of established tumors, with no significant difference between them. Thus, the Listeria vaccine vector presented here potentially complies with Food and Drug Administration regulations and could be developed further for clinical use.
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Wang, Yufu, Changcheng You, Rongzhi Wei, Jianing Zu, Chengchao Song, Jing Li, and Jinglong Yan. "Modification of Human Umbilical Cord Blood Stem Cells Using Polyethylenimine Combined with Modified TAT Peptide to Enhance BMP-2 Production." BioMed Research International 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/2971413.
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With the emerging role of umbilical cord blood-derived mesenchymal stem cells (hUCB-MSC) for bone regeneration and delivery of therapeutic proteins, there is an increasing need for effective gene delivery systems to modify such cells. mTAT, a TAT peptide sequence bearing histidine and cysteine residues, has been successfully used for intracellular gene delivery. Using a gWiz-GFP plasmid, we demonstrated that polyethylenimine combined with mTAT (mTAT/PEI) displayed good transfection efficacy in hUCB-MSC. hUCB-MSC transfected with mTAT/PEI were shown to express more BMP-2 protein and mRNA, indicating the feasibility of using the cells as a BMP-2 delivery system. Importantly, compared to PEI25, a “gold standard” nonviral transfection polymer, mTAT/PEI had limited toxicity to the cells. Furthermore, we demonstrated enhanced osteogenic activity in vitro for BMP-2 expressing hUCB-MSC. These results provide encouraging evidence for the potential use of mTAT/PEI to genetically modify hUCB-MSC as an approach to enhance tissue regeneration.
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Glenting, Jacob, Søren M. Madsen, Astrid Vrang, Anders Fomsgaard, and Hans Israelsen. "A Plasmid Selection System in Lactococcus lactis and Its Use for Gene Expression in L. lactis and Human Kidney Fibroblasts." Applied and Environmental Microbiology 68, no. 10 (October 2002): 5051–56. http://dx.doi.org/10.1128/aem.68.10.5051-5056.2002.
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ABSTRACT We report the development of a nonantibiotic and nonpathogenic host-plasmid selection system based on lactococcal genes and threonine complementation. We constructed an auxotrophic Lactococcus lactis MG1363Δthr strain which carries deletions in two genes encoding threonine biosynthetic enzymes. To achieve plasmid-borne complementation, we then constructed the minimal cloning vector, pJAG5, based on the genes encoding homoserine dehydrogenase-homoserine kinase (the hom-thrB operon) as a selective marker. Using strain MG1363Δthr, selection and maintenance of cells carrying pJAG5 were obtained in threonine-free defined media. Compared to the commonly used selection system based on erythromycin resistance, the designed complementation system offers a competitive and stable plasmid selection system for the production of heterologous proteins in L. lactis. The potential of pJAG5 to deliver genes for expression in eukaryotes was evaluated by insertion of a mammalian expression unit encoding a modified green fluorescent protein. The successful delivery and expression of genes in human kidney fibroblasts indicated the potential of the designed nonantibiotic host-plasmid system for use in genetic immunization.
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Gollan, Timothy J., and Michael R. Green. "Redirecting Retroviral Tropism by Insertion of Short, Nondisruptive Peptide Ligands into Envelope." Journal of Virology 76, no. 7 (April 1, 2002): 3558–63. http://dx.doi.org/10.1128/jvi.76.7.3558-3563.2002.
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ABSTRACT A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.
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de Jonge, Jørgen, Johanna M. Leenhouts, Marijke Holtrop, Pieter Schoen, Peter Scherrer, Pieter R. Cullis, Jan Wilschut, and Anke Huckriede. "Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA." Biochemical Journal 405, no. 1 (June 13, 2007): 41–49. http://dx.doi.org/10.1042/bj20061756.
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Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.
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Jacobs, Liesl, Elien De Smidt, Nick Geukens, Kevin Hollevoet, and Paul Declerck. "586 Intratumoral DNA-based gene transfer as an efficient delivery approach to combine checkpoint-inhibiting antibodies with interleukin 12." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A621. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0586.
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BackgroundCheckpoint inhibitors have demonstrated clinical benefit for several types of cancer, but still a large proportion of patients do not respond to treatment. To improve response rates, many combination therapies are currently under clinical evaluation. One such example is the combination of anti-PD-1 monoclonal antibodies with intratumoral gene transfer of plasmid-based interleukin 12 (IL-12). Local expression of the cytokine IL-12 has been shown to increase immune cell infiltration in cold tumors, which can make them more responsive to anti-PD-1 antibodies.1 The current study evaluates the efficacy of simultaneous delivery of checkpoint-inhibiting antibodies and IL-12 by intratumoral gene transfer. We recently demonstrated that intratumoral delivery of plasmid-based checkpoint inhibitors yielded systemic anti-tumor responses in a mouse tumor model, with only limited systemic antibody exposure and therefore improved biosafety.2MethodsC57BL/6J mice bearing a subcutaneous syngeneic MC38 tumor received a single intratumoral injection of plasmid DNA followed by in vivo electroporation. DNA-based IL-12 (p(IL-12), 2.5 µg) was administered alone or in combination with a DNA-based anti-PD-1 antibody (p(aPD-1), 60 µg) and/or DNA-based anti-CTLA-4 antibody (p(aCTLA-4), 60 µg). Abscopal effects were studied in mice bearing two contralateral tumors, of which only one received therapy.ResultsThe combined intratumoral delivery of p(IL-12) and p(aPD-1) resulted in 10% complete responders, in contrast to no complete tumor regressions with each individual treatment. Yet, differences in tumor growth or survival did not reach statistical significance between these groups. To improve anti-tumor efficacy, the combined gene transfer was expanded with a second DNA-based checkpoint inhibitor, p(aCTLA-4). While intratumoral delivery of this triple combination also led to 10% complete regressions, the response did result in significant tumor growth delay compared to p(IL-12) alone (p<0.05) and the combination of both checkpoint inhibitors (p<0.01). Moreover, in a dual MC38 tumor model, the triple combination enabled significant abscopal effects compared to untreated mice (p<0.01), which was not the case for the other treatments.ConclusionsThis study demonstrates that intratumoral DNA-based gene transfer can be applied to efficiently combine different immunotherapeutics. This approach allows simplification of the treatment schedule, addresses the complex production of conventional protein-based therapeutics, and enables local drug expression, thereby minimizing systemic exposure and subsequent adverse events. Ongoing studies focus on the further validation of combined intratumoral delivery of plasmid-based checkpoint inhibitors and IL-12, by investigating the effect on tumor-infiltrating and peripheral immune cells as well as through evaluation of the triple combination in other tumor models.Ethics ApprovalThis study was approved by the KU Leuven Animal Ethics Committee, approval number P130/2017.ReferencesAlgazi AP, et al. Phase II Trial of IL-12 Plasmid Transfection and PD-1 Blockade in Immunologically Quiescent Melanoma. Clin Cancer Res 2020; 26:2827–2837.Jacobs L, et al. DNA-based delivery of checkpoint inhibitors in muscle and tumor enables long-term responses with distinct exposure. Mol Ther 2020;28:1068–1077.
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Akbaba, Hasan, Gulsah Erel-Akbaba, and Serif Senturk. "Special Focus Issue Part II: Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing." Nanomedicine 16, no. 12 (May 2021): 963–78. http://dx.doi.org/10.2217/nnm-2020-0412.
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Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.
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Qian, Feng, and Weiqing Pan. "Construction of a tetR-Integrated Salmonella enterica Serovar Typhi CVD908 Strain That Tightly Controls Expression of the Major Merozoite Surface Protein of Plasmodium falciparum for Applications in Human Vaccine Production." Infection and Immunity 70, no. 4 (April 2002): 2029–38. http://dx.doi.org/10.1128/iai.70.4.2029-2038.2002.
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ABSTRACT Attenuated Salmonella strains are an attractive live vector for delivery of a foreign antigen to the human immune system. However, the problem with this vector lies with plasmid segregation and the low level of expression of the foreign gene in vivo when constitutive expression is employed, leading to a diminished immune response. We have established inducible expressions of foreign genes in the Salmonella enterica serovar Typhi CVD908 vaccine strain using the tetracycline response regulatory promoter. To set up this system, a tetracycline repressor (tetR) was integrated into a defined ΔaroC locus of the chromosome via suicide plasmid pJG12/tetR-neo. To remove the neo gene conferring kanamycin resistance from the locus, a cre expression vector under the control of the tetracycline response promoter was transformed into the clone; expression of the Cre recombinase excised the neo gene and generated the end strain CVD908-tetR. Expression of the luciferase reporter gene in this strain is dependent on the presence of tetracycline in the medium and can be regulated up to 4,773-fold. Moreover, the tightly controlled expression of major merozoite surface protein 1 (MSP1) and parts of Plasmodium falciparum was achieved, and the product yield was increased when the inducible expression system was employed. Inoculation of bacteria harboring plasmid pZE11/MSP142 in mice produced the protein in liver and spleen controlled by the inducer. The persistence of the plasmid-carrying bacteria in mice was determined. Peak colonization of both liver and spleen was detected on the third day postinoculation and was followed by a decline in growth curves. After 14 days postinfection, the majority of the bacteria (>90%) recovered from the liver and spleen of the mice retained the plasmid when expression was induced; this clearly indicated that stability of the expression vector in vivo was improved by inducible expression. Establishment of the regulatory system in the vaccine strain may broaden the range of its use by enhancing plasmid stability and expression levels in vivo. Moreover, the availability of the vaccine strain inducibly expressing the entire MSP1 provides possibilities for examining its immunogenicity, particularly the cellular response in animal models.
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Nagahara, Shiori, Tetsuya Higashiyama, and Yoko Mizuta. "Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube." Plant Reproduction 34, no. 3 (June 19, 2021): 191–205. http://dx.doi.org/10.1007/s00497-021-00418-z.
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Abstract Key message Biolistic delivery into pollen. Abstract In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.
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MacColl, GS, FJ Novo, NJ Marshall, M. Waters, G. Goldspink, and PM Bouloux. "Optimisation of growth hormone production by muscle cells using plasmid DNA." Journal of Endocrinology 165, no. 2 (May 1, 2000): 329–36. http://dx.doi.org/10.1677/joe.0.1650329.
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The production of peptide hormones by skeletal muscle tissue is a promising area of gene therapy. Skeletal muscle myogenesis can be induced in vitro, resulting in the fusion of mononucleate myoblasts to form multinucleate myotubes, and delivery vectors are first tested in vitro. C2C12 myoblasts transfected with pcDNA3-GH, which used the human cytomegalovirus (CMV) promoter, secreted immunoreactive GH with comparable biological activity to pituitary GH. Mouse myeloid leukaemia cells, which express the mouse GH receptor were used for the bioassay, and activation of these cells by GH was measured by a colorimetric microculture tetrazolium assay. Cells were incubated with a tetrazolium salt (MTS) and an intermediate electron acceptor (phenazine methosulphate, PMS), and formazan production was measured as optical density (O.D.) at 490 nm. The efficiencies of several plasmid expression vectors were compared in differentiated and non-differentiated muscle cells, as a function of bioactive GH secreted by the transfected cells. Ten-day differentiated C2C12 myotubes transfected with pcDNA3E-GH, which used the CMV promoter and a rat myosin light chain enhancer element, secreted significantly more biologically active GH than myotubes transfected with pcDNA3-GH (0.82 O.D. units+/-0.06 vs 0.57+/-0.05 respectively, P<0.001). This was consistent with reduced CMV promoter activity in myotubes. Myoblasts transfected with pcDNA3-GH secreted more bioactive GH than 10-day transfected myotubes (1.1+/-0. 1 vs 0.77+/-0.07 respectively). However, the responses were indistinguishable (both 1.0+/-0.09) if both the myotubes and myoblasts had been transfected with pcDNA3E-GH. Substitution of the vector pMHLC-GH, which used a muscle-specific truncated rabbit myosin heavy chain promoter, and the myosin enhancer resulted in a marked decrease in the responses to the conditioned medium from fused myotubes compared with the vectors pcDNA3-GH and pcDNA3E-GH (0. 24+/-0.02 vs 0.57+/-0.05 vs 0.82+/-0.06 respectively). We concluded that the combination of CMV promoter and myosin light chain enhancer in pcDNA3E-GH had the greatest expression efficiency of the several plasmid vectors which we investigated.
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Li, Song, Su-Ping Wu, Mark Whitmore, Eric J. Loeffert, Lin Wang, Simon C. Watkins, Bruce R. Pitt, and Leaf Huang. "Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors." American Journal of Physiology-Lung Cellular and Molecular Physiology 276, no. 5 (May 1, 1999): L796—L804. http://dx.doi.org/10.1152/ajplung.1999.276.5.l796.
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Cationic lipid-mediated intravenous gene delivery shows promise in treating pulmonary diseases including lung tumor metastases, pulmonary hypertension, and acute respiratory distress syndrome. Nevertheless, clinical applications of cationic lipidic vectors via intravenous administration are limited by their transient gene expression. In addition, repeated dosing is not effective at frequent intervals. In an effort to elucidate the mechanism of gene inactivation, we report in this study that cationic lipid-protamine-DNA (LPD) complexes, but not each component alone, can induce a high level of cytokine production, including interferon-γ and tumor necrosis factor-α. Furthermore, we demonstrate that LPD administration triggers apoptosis in the lung, a phenomenon that may be mediated in part by the two cytokines. Treatment of mice with antibodies against the two cytokines prolongs the duration of gene expression and also improves lung transfection on a second administration of LPD. Although the mechanism underlying LPD-induced cytokine production is unclear, methylation of the DNA significantly decreased the level of both interferon-γ and tumor necrosis factor-α, suggesting that unmethylated CpG sequences in plasmid DNA play an important role. These data suggest that decreasing the CpG-mediated immune response while not affecting gene expression may be a useful therapeutic strategy to improve cationic lipid-mediated intravenous gene delivery to the lung.
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Bergquist, P. L., V. S. J. Te'o, M. D. Gibbs, N. C. Curach, and K. M. H. Nevalainen. "Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts." Biochemical Society Transactions 32, no. 2 (April 1, 2004): 293–97. http://dx.doi.org/10.1042/bst0320293.
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Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical. We have tested two fungal systems for the bulk production of enzymes from thermophiles. The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter and recombinant plasmid DNAs introduced into various high-secreting T. reesei strains using biolistic particle delivery. In some cases (e.g. the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes. The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains. A proteomics approach has been taken to identify strongly expressed proteins produced by T. reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins. Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions. The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium.
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Neves, Ana Raquel, Tânia Albuquerque, Rúben Faria, Milan Paul, Swati Biswas, Ângela Sousa, and Diana Costa. "Development of Tailor-Made Dendrimer Ternary Complexes for Drug/Gene Co-Delivery in Cancer." Pharmaceutics 13, no. 8 (August 13, 2021): 1256. http://dx.doi.org/10.3390/pharmaceutics13081256.
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Cancer gene therapy, mediated by non-viral systems, remains a major research focus. To contribute to this field, in this work we reported on the development of dendrimer drug/gene ternary complexes. This innovative approach explored the great capacity of both polyamidoamine (PAMAM)-paclitaxel (PTX) conjugate and polyethylenimine (PEI) polymers to complex a p53-encoding plasmid DNA (pDNA), highlighting the utility of considering two compacting agents. The pDNA complexation capacity has been investigated as function of the nitrogen to phosphate groups ratio (N/P), which revealed to be a tailoring parameter. The physicochemical properties of the conceived ternary complexes were revealed and were found to be promising for cellular transfection. Furthermore, the formulated co-delivery systems demonstrated to be biocompatible. The ternary systems were able of cellular internalization and payload intracellular release. Confocal microscopy studies showed the co-localization of stained pDNA with the nucleus of cancer cells, after transfection mediated by these carriers. From this achievement, p53 gene expression occurred with the production of protein. Moreover, the activation of caspase-3 indicated apoptosis of cancer cells. This work represents a great progress on the design of dendrimer drug/gene co-delivery systems towards a more efficient cancer therapy. In this way, it instigates further in vitro studies concerning the evaluation of their therapeutic potential, expectedly supported by the synergistic effect, in tumoral cells.
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Srinivasakumar, Narasimhachar, and Friedrich G. Schuening. "A Lentivirus Packaging System Based on Alternative RNA Transport Mechanisms To Express Helper and Gene Transfer Vector RNAs and Its Use To Study the Requirement of Accessory Proteins for Particle Formation and Gene Delivery." Journal of Virology 73, no. 11 (November 1, 1999): 9589–98. http://dx.doi.org/10.1128/jvi.73.11.9589-9598.1999.
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ABSTRACT A lentivirus-based packaging system was designed to reduce the chance of recombination between helper and gene transfer vector sequences by using the constitutive transport element (CTE) derived from Mason-Pfizer monkey virus for expression of the viral proteins and the Rev-Rev response element (RRE) combination for expression of the gene transfer vector. Using this approach, we evaluated a series of human immunodeficiency virus type 1 packaging constructs that express one or more accessory proteins (Vif, Vpr, and Vpu), in addition to the Gag and Pol proteins, for particle formation and virus stock production for gene transfer. Constructs that also express Vpr or both Vpr and Vpu produced more particles, as measured by a p24 assay, than did plasmids that did not contain these sequences. Transactivation experiments showed that the packaging plasmids that encode Vpr or both Vpr and Vpu also expressed a functional single-exon Tat protein. For these constructs, high-titer virus stocks could be prepared in the absence of a cotransfected Tat-expressing plasmid. Amphotropic-envelope-pseudotyped virus stocks prepared with all of the packaging constructs, irrespective of whether any of the accessory proteins were coexpressed, were equally efficient in transducing growth-arrested HeLa cells. The combination/mixed packaging system was compared to systems that were based on either the CTE alone or Rev and RRE for expression of both the packaging plasmid as well as the gene transfer vector. The combination/mixed packaging system was comparable to the other systems for production of virus stocks, suggesting that this design may prove to be safer for the eventual deployment of lentivirus vectors for therapeutic purposes.
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Ye, Peiqing, and Carol H. Miao. "Dependence of Vector Dose and Age of Mice in the Induction of Immune Response Against Factor VIII Following Liver-Directed Nonviral Gene Transfer." Blood 104, no. 11 (November 16, 2004): 3186. http://dx.doi.org/10.1182/blood.v104.11.3186.3186.
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Abstract Formation of inhibitory antibodies to transgene product may limit the success of gene therapy especially for the treatment of hemophilia A. The risk of forming inhibitory antibodies against factor VIII depends on multiple factors. Previously we have shown that following naked gene transfer of fifty micrograms of a liver-specific, high-expressing factor VIII plasmid, pBS-HCRHPI-FVIIIA into hemophilia A mice (at least 60 days old), a robust humoral response was induced two weeks post plasmid injection despite of initial high-level gene expression of factor VIII (Ye et al. (2004) Mol. Ther. 10, 117–126). This response completely inhibited the activity of circulating factor VIII although factor VIII was persistently expressed in the liver. In this study, the cytokine production was characterized in human factor VIII-activated T cells from plasmid-treated and untreated hemophilic A mice, consistent with a response predominantly mediated by Th2-induced signals. Injection of plasmid DNA into 4 groups of hemophilia A mice (n=5, 60 days old) with 4 different doses (0.4, 2, 10, & 50 microgram per animal) resulted in vector dose-dependent expression of factor VIII. In addition, the two groups of mice with lower doses of plasmid DNA (0.4 & 2 microgram per animal) did not elicit any antibody response against factor VIII, whereas the two groups of mice with higher doses of plasmid DNA (10 & 50 microgram per animal) induced inhibitory antibody formation. Nevertheless, when the two groups of animals (n=4) with lower doses were treated with second injection of fifty microgram of factor VIII plasmid 180 days post plasmid delivery, all mice developed inhibitors suggesting no immune tolerance was induced by first injection of plasmids. Furthermore, fifty micrograms of factor VIII plasmids were injected into 4 groups of hemophilia A mice (n=5) of 4 different age groups (36, 48, 60 & 72 days). It was found that none of the mice with age 36 days at the time of plasmid injection developed inhibitors, 1/5 mice with age 48 days developed inhibitors, whereas the two groups of mice with age 60 & 72 days all developed high-titer inhibitors. These results indicate that induction of anti-factor VIII antibody following gene therapy is strongly dependent upon the vector dose and age of the animals, which has important implication for developing immunomodulation or other strategies to avoid/eliminate antibody responses.
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Cai, Yujia, Rasmus O. Bak, Louise Bechmann Krogh, Nicklas H. Staunstrup, Brian Moldt, Thomas J. Corydon, Lisbeth Dahl Schrøder, and Jacob Giehm Mikkelsen. "DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors." Nucleic Acids Research 42, no. 4 (November 21, 2013): e28-e28. http://dx.doi.org/10.1093/nar/gkt1163.
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Abstract DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.
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Zou, Wei, Fang Cheng, Weiran Shen, John F. Engelhardt, Ziying Yan, and Jianming Qiu. "Nonstructural Protein NP1 of Human Bocavirus 1 Plays a Critical Role in the Expression of Viral Capsid Proteins." Journal of Virology 90, no. 9 (February 24, 2016): 4658–69. http://dx.doi.org/10.1128/jvi.02964-15.
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ABSTRACTA novel chimeric parvoviral vector, rAAV2/HBoV1, in which the recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged by the human bocavirus 1 (HBoV1) capsid, has been shown to be highly efficient in gene delivery to human airway epithelia (Z. Yan et al., Mol Ther 21:2181–2194, 2013,http://dx.doi.org/10.1038/mt.2013.92). In this vector production system, we used an HBoV1 packaging plasmid, pHBoV1NSCap, that harbors HBoV1 nonstructural protein (NS) and capsid protein (Cap) genes. In order to simplify this packaging plasmid, we investigated the involvement of the HBoV1 NS proteins in capsid protein expression. We found that NP1, a small NS protein encoded by the middle open reading frame, is required for the expression of the viral capsid proteins (VP1, VP2, and VP3). We also found that the other NS proteins (NS1, NS2, NS3, and NS4) are not required for the expression of VP proteins. We performed systematic analyses of the HBoV1 mRNAs transcribed from the pHBoV1NSCap packaging plasmid and its derivatives in HEK 293 cells. Mechanistically, we found that NP1 is required for both the splicing and the read-through of the proximal polyadenylation site of the HBoV1 precursor mRNA, essential functions for the maturation of capsid protein-encoding mRNA. Thus, our study provides a unique example of how a small viral nonstructural protein facilitates the multifaceted regulation of capsid gene expression.IMPORTANCEA novel chimeric parvoviral vector, rAAV2/HBoV1, expressing a full-length cystic fibrosis transmembrane conductance regulator (CFTR) gene, is capable of correcting CFTR-dependent chloride transport in cystic fibrosis human airway epithelium. Previously, an HBoV1 nonstructural and capsid protein-expressing plasmid, pHBoV1NSCap, was used to package the rAAV2/HBoV1 vector, but yields remained low. In this study, we demonstrated that the nonstructural protein NP1 is required for the expression of capsid proteins. However, we found that the other four nonstructural proteins (NS1 to -4) are not required for expression of capsid proteins. By mutating theciselements that function as internal polyadenylation signals in the capsid protein-expressing mRNA, we constructed a simple HBoV1 capsid protein-expressing gene that expresses capsid proteins as efficiently as pHBoV1NSCap does, and at similar ratios, but independently of NP1. Our study provides a foundation to develop a better packaging system for rAAV2/HBoV1 vector production.
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Zheng, Feiyang, Yoshinori Kawabe, Mai Murakami, Mamika Takahashi, Shoichiro Yoshida, Akira Ito, and Masamichi Kamihira. "Retrotransposon-mediated Gene Transfer for Animal Cells." MATEC Web of Conferences 333 (2021): 07002. http://dx.doi.org/10.1051/matecconf/202133307002.
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Gene delivery methods for animal cells are one of the most important tools in biotechnology fields such as pharmaceutical protein production, generation of transgenic animals and gene therapy. Because retrotransposons can move their own sequences to new genomic locations by a “copy-and-paste” process known as retrotransposition, we attempted to develop a novel gene transfer system based on retrotransposon. A full-length long interspersed element-1 (LINE-1) contains a 5’ untranslated region (5’UTR), two non-overlapping open reading frames (ORFs) separated by a short inter-ORF sequence, and a 3’UTR terminating in an adenosine-rich tract. We constructed a LINE-1 vector plasmid including components necessary for retrotransposition. An intron-disrupted Neo reporter gene and a scFv-Fc expression unit under the control of CMV promoter were added into 3’UTR in order to evaluate retrotransposition and express scFv-Fc. CHO-K1 cells transfected with the plasmids were screened with G418. The established cell clones produced scFv-Fc proteins in the culture medium. To control retrotransposition steadily, we also established retrotransposon systems that supply ORF2 or ORF1–2 separately. Genomic PCR analysis revealed that transgene sequences derived from the LINE-1 vector were positive in all clones. All the clones tested produced scFv-Fc in the culture medium.
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Zheng, Feiyang, Yoshinori Kawabe, Mai Murakami, Mamika Takahashi, Shoichiro Yoshida, Akira Ito, and Masamichi Kamihira. "Retrotransposon-mediated Gene Transfer for Animal Cells." MATEC Web of Conferences 333 (2021): 07002. http://dx.doi.org/10.1051/matecconf/202133307002.
Full textAbstract:
Gene delivery methods for animal cells are one of the most important tools in biotechnology fields such as pharmaceutical protein production, generation of transgenic animals and gene therapy. Because retrotransposons can move their own sequences to new genomic locations by a “copy-and-paste” process known as retrotransposition, we attempted to develop a novel gene transfer system based on retrotransposon. A full-length long interspersed element-1 (LINE-1) contains a 5’ untranslated region (5’UTR), two non-overlapping open reading frames (ORFs) separated by a short inter-ORF sequence, and a 3’UTR terminating in an adenosine-rich tract. We constructed a LINE-1 vector plasmid including components necessary for retrotransposition. An intron-disrupted Neo reporter gene and a scFv-Fc expression unit under the control of CMV promoter were added into 3’UTR in order to evaluate retrotransposition and express scFv-Fc. CHO-K1 cells transfected with the plasmids were screened with G418. The established cell clones produced scFv-Fc proteins in the culture medium. To control retrotransposition steadily, we also established retrotransposon systems that supply ORF2 or ORF1–2 separately. Genomic PCR analysis revealed that transgene sequences derived from the LINE-1 vector were positive in all clones. All the clones tested produced scFv-Fc in the culture medium.
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Pergolizzi, Robert G., Rachel Dragos, Diane Chan, Denisa D. Wagner, Peter J. Lenting, and Ronald G. Crystal. "Correction of a Murine Model of Von Willebrand Disease by Gene Transfer." Blood 104, no. 11 (November 16, 2004): 3180. http://dx.doi.org/10.1182/blood.v104.11.3180.3180.
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Abstract Von Willebrand disease (VWD) is the most common inherited bleeding disorder affecting 1 to 3% of the U.S. population. It is caused by defects in expression and processing of Von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis, that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. Since it is established that 1–5% of normal levels of VWF is sufficient to ameliorate the bleeding phenotype, the possibility that corrective levels of circulating VWF could be produced using a gene transfer strategy was investigated. However, since the normal sites of VWF production, endothelial cells, megakaryocytes and platelets, are difficult to target with traditional gene transfer methodologies, we explored the strategy of using gene transfer to the liver, an easily transduced ectopic site for VWF production. The study employed a murine “knockout” model of VWD created by disrupting the naturally occurring VWF gene (VWF-KO mice) which exhibit defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events closely mimicking severe human VWD. Transected-tail wounds in untreated VWF-KO mice will bleed to death unless cauterized, whereas similar wounds in WT mice stop bleeding in <3 min. To investigate the impact of VWF gene transfer on this phenotype, a plasmid containing the complete murine VWF cDNA under the control of a cytomegalovirus immediate/early promoter/enhancer, was delivered to the VWF-KO mice intravenously. An identical plasmid containing the cDNA for green fluorescent protein (GFP) and saline were used as controls. Bleeding time after wounding was determined 24, 48 or 72 hr after transfer of 10 to 250 μg of plasmid. Bleeding time was normalized by 48 hr after delivery of 50 μg or more of VWF cDNA, but not by GFP cDNA. Western analysis of plasma from animals receiving VWF cDNA, but not GFP cDNA, revealed high-molecular weight VWF multimers similar to the pattern observed in wild type mice. These data suggest that it may be possible to correct VWD using gene therapy delivered to tissues other than the naturally occurring sites of VWF manufacture, and that the liver is capable of expressing, processing and secreting VWF in a physiologically active form.
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Selinger, L. B., G. G. Khachatourians, J. R. Byers, and M. F. Hynes. "Expression of a Bacillus thuringiensis δ-endotoxin gene by Bacillus pumilus." Canadian Journal of Microbiology 44, no. 3 (March 1, 1998): 259–69. http://dx.doi.org/10.1139/w97-147.
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The δ -endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a δ -endotoxin expression and delivery system for subterranean feeding insects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pale western cutworm larvae. Three different cry genes from Bacillus thuringiensis subsp. kurstaki were cloned into high and low copy number vectors and mated into Bacillus pumilus RB8. When carried on high copy number vectors, cry genes appeared to inhibit sporulation and δ -endotoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that <0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal inclusions. On low copy number vectors, the cry genes did not inhibit sporulation; however, production of δ -endotoxins was undetectable. Using a heat shock regime for enrichment of sporogenous crystalliferous variants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid, was obtained in which high level δ -endotoxin production occurred concomitant with sporulation. Synthesis of functional δ -endotoxin by this strain was confirmed by Western blot analysis and bioassay with pale western cutworm larvae. These results show that rhizosphere-inhabiting bacilli are indeed a potential route for introduction of δ -endotoxins to the root environment for biocontrol purposes.Key words: Bacillus thuringiensis, δ -endotoxin, conjugation, sporulation, expression.
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Kajikawa, Akinobu, Kazuya Masuda, Mitsunori Katoh, and Shizunobu Igimi. "Adjuvant Effects for Oral Immunization Provided by Recombinant Lactobacillus casei Secreting Biologically Active Murine Interleukin-1β." Clinical and Vaccine Immunology 17, no. 1 (November 18, 2009): 43–48. http://dx.doi.org/10.1128/cvi.00337-09.
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ABSTRACT Vaccine delivery systems using lactic acid bacteria are under development, but their efficiency is insufficient. Autologous cytokines, such as interleukin-1β (IL-1β), are potential adjuvants for mucosal vaccines and can be provided by recombinant lactic acid bacteria. The aim of this study was the construction and evaluation of recombinant Lactobacillus casei producing IL-1β as an adjuvant delivery agent. The recombinant strain was constructed using an expression/secretion vector plasmid, including a mature IL-1β gene from mouse. The biological activity of the cytokine was confirmed by IL-8 production from Caco-2 cells. In response to the recombinant L. casei secreting IL-1β, expression of IL-6 was detected in vivo using a ligated-intestinal-loop assay. The release of IL-6 from Peyer's patch cells was also detected in vitro. Intragastric immunization with heat-killed Salmonella enterica serovar Enteritidis (SE) in combination with IL-1β-secreting lactobacilli resulted in relatively high SE-specific antibody production. In this study, it was demonstrated that recombinant L. casei secreting bioactive murine IL-1β provided adjuvant effects for intragastric immunization.
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Ferraro, B., K. T. Talbott, A. Balakrishnan, N. Cisper, M. P. Morrow, N. A. Hutnick, D. J. Myles, et al. "Inducing Humoral and Cellular Responses to Multiple Sporozoite and Liver-Stage Malaria Antigens Using Exogenous Plasmid DNA." Infection and Immunity 81, no. 10 (July 29, 2013): 3709–20. http://dx.doi.org/10.1128/iai.00180-13.
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ABSTRACTA vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection againstPlasmodium falciparummalaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets fourP. falciparumantigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered usingin vivoelectroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4+and CD8+T cell compartments. Furthermore, hepatic CD8+lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8+granzyme B+T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.
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Ohlfest, John R., Joel L. Frandsen, Sabine Fritz, Paul D. Lobitz, Scott G. Perkinson, Karl J. Clark, Gary Nelsestuen, et al. "Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system." Blood 105, no. 7 (April 1, 2005): 2691–98. http://dx.doi.org/10.1182/blood-2004-09-3496.
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AbstractHemophilia A is a lead candidate for treatment by gene therapy because small increments in the missing secreted protein product, coagulation factor VIII (FVIII), would result in substantial clinical amelioration. Clinically relevant therapy might be achieved by stably delivering a human FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by tail-vein injection, to integrate B-domain–deleted FVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since FVIII protein is a neoantigen to these mice, sustaining therapeutic plasma FVIII levels was problematic due to inhibitory antibody production. We circumvented this problem by tolerizing 82% of neonates by a single facial-vein injection of recombinant FVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10%-100% of normal) FVIII expression and phenotypic correction required co-injection of an SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated polymerase chain reaction was used to clone FVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained FVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented.
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Bigot, Karine, Pauline Gondouin, Romain Bénard, Pierrick Montagne, Jenny Youale, Marie Piazza, Emilie Picard, Thierry Bordet, and Francine Behar-Cohen. "Transferrin Non-Viral Gene Therapy for Treatment of Retinal Degeneration." Pharmaceutics 12, no. 9 (September 1, 2020): 836. http://dx.doi.org/10.3390/pharmaceutics12090836.
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Dysregulation of iron metabolism is observed in animal models of retinitis pigmentosa (RP) and in patients with age-related macular degeneration (AMD), possibly contributing to oxidative damage of the retina. Transferrin (TF), an endogenous iron chelator, was proposed as a therapeutic candidate. Here, the efficacy of TF non-viral gene therapy based on the electrotransfection of pEYS611, a plasmid encoding human TF, into the ciliary muscle was evaluated in several rat models of retinal degeneration. pEYS611 administration allowed for the sustained intraocular production of TF for at least 3 and 6 months in rats and rabbits, respectively. In the photo-oxidative damage model, pEYS611 protected both retinal structure and function more efficiently than carnosic acid, a natural antioxidant, reduced microglial infiltration in the outer retina and preserved the integrity of the outer retinal barrier. pEYS611 also protected photoreceptors from N-methyl-N-nitrosourea-induced apoptosis. Finally, pEYS611 delayed structural and functional degeneration in the RCS rat model of RP while malondialdehyde (MDA) ocular content, a biomarker of oxidative stress, was decreased. The neuroprotective benefits of TF non-viral gene delivery in retinal degenerative disease models further validates iron overload as a therapeutic target and supports the continued development of pEY611 for treatment of RP and dry AMD.
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Al-Ubaidi, Ghassaq T., Ahmed A. Abbas, Ali A. Taha, Qasim S. Sharhan, Israa W. Ahmed, and Ilham A. Jasim. "Evaluation of Synergistic Effect of Cytosine-Phosphodiesterbond-Guanosin-Oligodeoxynucleotide 7909, and Protamine on Transfection Process Mediated by Calcium Phosphate Nanoparticles." International Journal of Drug Delivery Technology 10, no. 01 (March 25, 2020): 52–59. http://dx.doi.org/10.25258/ijddt.10.1.9.
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Bacille Calmette-Guerin (BCG) still the only authenticated vaccine against tuberculosis. Due to its drawbacks, a need for a new formula has emerged. The implication of “Nanovaccinology” is one of the possible alternatives. The non-viral vectors have a low transfection ability. In the context, this work aims to add two adjuvants to a calcium phosphate nanoparticles (CPNPs) functionalized with early secreted antigenic target 6-kilo dalton ( ESAT-6) cloned pcDNA3.1(+) plasmid. ESAT-6 gene is specific to mycobacterium tuberculosis complex (MTC) and encodes a T-cell antigen. The adjuvants in practice are Herring protamine and cytosine-phosphodiester bond-guanosine-oligodeoxynucleotide 7909 ( CpG-ODN 7909). Each has a different strategy in enhancing immune response; protamine is particulate adjuvant while CpG is an immunopotentiator substance. Nano complex was transfected into THP-1 monocytic cell line after its activation to a macrophage via 100nM PMA. Cellular immune response, interleukin-12 (IL-12), and tumor necrosis factor –alfa (TNF-ɑ) also ESAT-6 protein production were assayed via the Sandwich ELISA technique. Results revealed that CPNPs offer only partial protection to the adsorbed plasmid against enzymatic degradation. Nano complex formula with two adjuvants resulted in significantly higher cellular immune response comparing to formula carrying one adjuvant. In conclusion, the implication of CPNPs in gene delivery accompanied with two adjuvants each possess different strategy, will result in partial protection to the delivered gene with upsurge cellular immune response.
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Muñoz-Montesino, Carola, Edilia Andrews, Rodolfo Rivers, Andrés González-Smith, Gustavo Moraga-Cid, Hugo Folch, Sandra Céspedes, and Angel A. Oñate. "Intraspleen Delivery of a DNA Vaccine Coding for Superoxide Dismutase (SOD) of Brucella abortus Induces SOD-Specific CD4+ and CD8+ T Cells." Infection and Immunity 72, no. 4 (April 2004): 2081–87. http://dx.doi.org/10.1128/iai.72.4.2081-2087.2004.
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ABSTRACT In the development of vaccines capable of providing immunity against brucellosis, Cu-Zn superoxide dismutase (SOD) has been demonstrated to be one of the protective immunogens of Brucella abortus. In an earlier study, we provided strong evidence that intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response. The present study was designed to characterize T-cell immune responses after an intraspleen (i.s.) vaccination of BALB/c mice with pcDNA-SOD. Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crude Brucella protein induced the secretion of gamma interferon (IFN-γ), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4+- and CD8+-T-cell populations. However, only the CD4+ population was able to produce IFN-γ and only the CD8+ population was able to induce cytotoxic activity. Nevertheless, although i.s. route vaccination induces a significant level of protection in BALB/c mice against challenge with the virulent B. abortus strain 2308, vaccination by the intramuscular route with a similar amount of plasmid DNA does not protect. Based on these results, we conclude that i.s. immunization with pcDNA-SOD vaccine efficiently induced a Th1 type of immune response and a protective response that could be related to IFN-γ production and cytotoxic activity against infected cells by SOD-specific CD4+ and CD8+ T cells, respectively.
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Rodolfo, Carlos, Dalinda Eusébio, Cathy Ventura, Renato Nunes, Helena F. Florindo, Diana Costa, and Ângela Sousa. "Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System." Pharmaceutics 13, no. 9 (August 31, 2021): 1369. http://dx.doi.org/10.3390/pharmaceutics13091369.
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In current times, DNA vaccines are seen as a promising approach to treat and prevent diseases, such as virus infections and cancer. Aiming at the production of a functional and effective plasmid DNA (pDNA) delivery system, four chitosan polymers, differing in the molecular weight, were studied using the design of experiments (DoE) tool. These gene delivery systems were formulated by ionotropic gelation and exploring the chitosan and TPP concentrations as DoE inputs to maximize the nanoparticle positive charge and minimize their size and polydispersity index (PDI) as DoE outputs. The obtained linear and quadratic models were statistically significant (p-value < 0.05) and non-significant lack of fit, with suitable coefficient of determination and the respective optimal points successfully validated. Furthermore, morphology, stability and cytotoxicity assays were performed to evaluate the endurance of these systems over time and their further potential for future in vitro studies. The subsequent optimization process was successful achieved for the delivery systems based on the four chitosan polymers, in which the smallest particle size was obtained for the carrier containing the 5 kDa chitosan (~82 nm), while the nanosystem prepared with the high molecular weight (HMW) chitosan displayed the highest zeta potential (~+26.8 mV). Delivery systems were stable in the formulation buffer after a month and did not exhibit toxicity for the cells. In this sense, DoE revealed to be a powerful tool to explore and tailor the characteristics of chitosan/pDNA nanosystems significantly contributing to unraveling an optimum carrier for advancing the DNA vaccines delivery field.
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Chey, Soroth, Juliane Maria Palmer, Laura Doerr, and Uwe Gerd Liebert. "Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells." Viruses 13, no. 9 (August 30, 2021): 1723. http://dx.doi.org/10.3390/v13091723.
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Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.
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Ando, Hidenori, Noriko Saito-Tarashima, Amr S. Abu Lila, Nozomi Kinjo, Taro Shimizu, Yu Ishima, Noriaki Minakawa, and Tatsuhiro Ishida. "A Unique Gene-Silencing Approach, Using an Intelligent RNA Expression Device (iRed), Results in Minimal Immune Stimulation When Given by Local Intrapleural Injection in Malignant Pleural Mesothelioma." Molecules 25, no. 7 (April 9, 2020): 1725. http://dx.doi.org/10.3390/molecules25071725.
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Background: We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. Methods: To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. Results: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Conclusion: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers.
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Qiao, Xi, Rong-Shan Li, Hong Li, Guo-Zhen Zhu, Xiao-Guang Huang, Shan Shao, and Bo Bai. "Intermedin protects against renal ischemia-reperfusion injury by inhibition of oxidative stress." American Journal of Physiology-Renal Physiology 304, no. 1 (January 1, 2013): F112—F119. http://dx.doi.org/10.1152/ajprenal.00054.2012.
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Reactive oxygen species (ROS) play a critical role in renal ischemia-reperfusion injury (IRI). Intermedin (IMD) reportedly protected against myocardial IRI via its antioxidant effects; however, its protective role in renal IRI has not been investigated. We overexpressed IMD in rat kidneys and examined how the kidneys respond to renal IRI. Eukaryotic expression plasmid encoding the rat IMD gene or control empty vector was transfected into the left kidney using an ultrasound-microbubble-mediated delivery system. This method yielded high expression of IMD in kidney cells. Renal IRI was induced by clamping the left renal artery followed by reperfusion. In response to IRI, overexpression of IMD in the kidney significantly improved renal function and pathology compared with the kidney transfected with control plasmid. We investigated the mechanisms by which IMD protects against renal IRI. We examined renal superoxide dismutase (SOD) activity and malondialdehyde (MDA) content and found SOD activity was significantly increased, while MDA level was markedly decreased in kidneys transfected with IMD, suggesting ROS production and oxidative stress were reduced by IMD overexpression. We also measured myeloperoxidase (MPO) activity, tubular cell apoptosis, and the expression of intercellular adhesion molecule-1 (ICAM-1), P-selectin, and endothelin-1 (ET-1) in the kidney. Renal MPO activity and the expression of ICAM-1, P-selectin, and ET-1 stimulated by IRI were significantly inhibited by IMD overexpression. Moreover, IMD overexpression prevented kidney cells from apoptosis caused by IRI. Our results demonstrate that overexpression of IMD in the kidney protects against renal IRI, apparently by reducing oxidative stress, consequently suppressing inflammation and vasoconstrictor production and apoptosis.
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Adel-Patient, Karine, Laetitia Pothelune, Sandrine Ah-Leung, Jean-Michel Wal, Christophe Créminon, and Jean-Marc Chatel. "Block Copolymers Have Differing Adjuvant Effects on the Primary Immune Response Elicited by Genetic Immunization and on Further Induced Allergy." Clinical and Vaccine Immunology 17, no. 1 (November 18, 2009): 36–42. http://dx.doi.org/10.1128/cvi.00275-09.
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ABSTRACT Block copolymers were recently used to promote gene delivery in various tissues. Using a plasmid encoding a food allergen, bovine β-lactoglobulin (BLG), we studied the effects of block copolymers on gene expression levels and primary immune response and on further induced allergy. Block copolymers (i.e., Tetronic 304, 908, and 1107) and various quantities of DNA were injected into the tibialis muscles of BALB/c mice. The BLG levels in injected muscle and the BLG-specific induced immune response were analyzed after injection. DNA-immunized mice were further experimentally sensitized with BLG, and the effects of block copolymer and DNA doses on allergic sensitization and elicitation were compared. Tetronic 304 induced a 12-fold increase in BLG production, while Tetronic 1107 increased the duration of BLG expression. Different Th1 primary specific immune responses were observed, either strong humoral and cellular (304), only cellular (1107), or weak cellular and humoral (908) responses. After BLG sensitization, increased BLG-specific IgG2a production was observed in all groups of mice independently of the presence and nature of the block copolymer. Increased BLG-specific IgG1 production was also detected after sensitization, except with Tetronic 1107. Compared with naked DNA, Tetronic 304 was the only block polymer that decreased BLG-specific IgE concentrations. However, after allergen challenge, Tetronic 1107 was the only block copolymer to reduce eosinophils and Th2 cytokines in bronchoalveolar lavage (BAL) fluid. Tetronic 304 amplified local inflammation. Each block copolymer elicited a different immune response, although always Th1 specific, in BALB/c mice.
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Tagliavia, Marcello, and Aldo Nicosia. "Advanced Strategies for Food-Grade Protein Production: A New E. coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expression." Microorganisms 7, no. 5 (April 27, 2019): 116. http://dx.doi.org/10.3390/microorganisms7050116.
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Food-grade production of recombinant proteins in Gram-positive bacteria, especially in LAB (i.e., Lactococcus, Lactobacillus, and Streptococcus), is of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene and metabolic engineering, as well as antigen delivery for oral vaccination. Food-grade expression relies on hosts generally considered as safe organisms and on clone selection not dependent on antibiotic markers, which limit the overall DNA manipulation workflow, as it can be carried out only in the expression host and not in E. coli. Moreover, many commercial expression vectors lack useful elements for protein purification. We constructed a “shuttle” vector containing a removable selective marker, which allows feasible cloning steps in E. coli and subsequent protein expression in LAB. In fact, the cassette can be easily excised from the selected recombinant plasmid, and the resulting marker-free vector transformed into the final LAB host. Further useful elements, as improved MCS, 6xHis-Tag, and thrombin cleavage site sequences were introduced. The resulting vector allows easy cloning in E. coli, can be quickly converted in a food-grade expression vector and harbors additional elements for improved recombinant protein purification. Overall, such features make the new vector an improved tool for food-grade expression.
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Lai, Erh-Min, and Clarence I. Kado. "Processed VirB2 Is the Major Subunit of the Promiscuous Pilus of Agrobacterium tumefaciens." Journal of Bacteriology 180, no. 10 (May 15, 1998): 2711–17. http://dx.doi.org/10.1128/jb.180.10.2711-2717.1998.
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ABSTRACT Previous studies have implicated the obligatory requirement for thevir regulon (or “virulon”) of the Ti plasmid for the transfer of oncogenes from Agrobacterium tumefaciens to plant cells. The machinery used in this horizontal gene transfer has been long thought to be a transformation or conjugative delivery system. Based on recent protein sequence comparisons, the proteins encoded by the virB operon are strikingly similar to proteins involved in the synthesis and assembly of conjugative pili such as the conjugative pilus of F plasmid in Escherichia coli. The F pilus is composed of TraA pilin subunits derived from TraA propilin. In the present study, evidence is provided showing that the counterpart of TraA is VirB2, which like TraA propilin is processed into a 7.2-kDa product that comprises the pilus subunit as demonstrated by biochemical and electron microscopic analyses. The processed VirB2 protein is present exocellularly on medium on which induced A. tumefaciens had grown and appears as thin filaments of 10 nm that react specifically to VirB2 antibody. Exocellular VirB2 is produced abundantly at 19°C as compared with 28°C, an observation that parallels the effect of low temperature on the production ofvir gene-specific pili observed previously (K. J. Fullner, L. C. Lara, and E. W. Nester, Science 273:1107–1109, 1996). Export of the processed VirB2 requires othervirB genes since mutations in these genes cause the loss of VirB2 pilus formation and result in processed VirB2 accumulation in the cell. The presence of exocellular processed VirB2 is directly correlated with the formation of pili, and it appears as the major protein in the purified pilus preparation. The evidence provides a compelling argument for VirB2 as the propilin whose 7.2-kDa processed product is the pilin subunit of the promiscuous conjugative pilus, hereafter called the “T pilus” of A. tumefaciens.
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Briski, O., G. La Motta, D. Salamone, R. Fernandez-Martin, and L. Ratner. "96 Evaluation of CRISPR/Cas9 alternative delivery in parthenogenetic porcine embryos." Reproduction, Fertility and Development 32, no. 2 (2020): 174. http://dx.doi.org/10.1071/rdv32n2ab96.
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Porcine genetic editing is considered promising in biomedical research, particularly for xenotransplantation. However, invitro porcine embryo production is less efficient than in other animal models. Thus, we aimed to perform a rapid optimization essay by producing parthenogenetic porcine embryos to evaluate transgenesis delivery and CRISPR/Cas9 editing efficiency. First, PCX-enhanced green fluorescent protein (EGFP) plasmid was microinjected (30ng μL−1) into a diploid parthenogenic zygote with (lipo+) or without lipofectamine (lipo−), since it has been shown that this transfection reagent improves transgene delivery and increases its expression in bovine embryos (Vichera et al. 2011 Reprod. Domest. Anim. 46, 214-220; https://doi.org/10.1111/j.1439-0531.2010.01642.x). Briefly, invitro-matured oocytes were electrically activated, followed by incubation in synthetic oviductal fluid medium containing 6-dimethylaminopyridine. Embryos were invitro cultured until Day 7, and EGFP-positive (EGFP+) embryos were corroborated at Day 5. Data were analysed using Fisher's exact test. Cleavage rates were no different among groups (EGFP/lipo−: 40%, n=28; EGFP/lipo+: 45%, n=41; control: 41, 3%, n=45; P>0.05). Also, no significant difference in the percentage of EGFP+ embryos was observed between EGFP/lipo+ (31%, n=13) and EGFP/Lipo− (18%, n=5) groups. Although blastocyst rate showed no statistical difference among groups, a lower blastocyst percentage tendency was observed in the EGFP/Lipo+ group compared with the control group (EGFP/Lipo+: 5%, n=2; control: 20%, n=9; P=0.051), suggesting that the presence of lipofectamine and EGFP plasmid may affect embryo development. Next, two guides (single guide (sg) RNA) were designed for the internal regions of GGTA1, CMAH, and VWF target genes, involved in hyperacute rejection and coagulation in xenotransplantation. A liposome-DNA mixture was used: DNA for sgRNA and Cas9, with and without lipofectamine (10× dilution; CRISPR/lipo+, CRISPR/lipo−), diluted to half concentration with 10% polyvinylpyrrolidone, resulting in a final concentration of 20ng ul−1 for all sgRNA and 40ng ul−1 for Cas9. A total of 2 pL of the mixtures was microinjected into the diploid parthenogenetic zygotes which were invitro cultured until Day 7. Genetic editing was corroborated by analysing the presence of a double cut directed by the two sgRNA designed for the target genes, resulting in an amplicon with lower molecular weight compared to the wild-type PCR fragment. No differences in cleavage or blastocyst rates were observed among groups (blastocyst rates: CRISPR/Lipo−: 12%, n=13; CRISPR/Lipo+: 10%, n=7; control: 15%, n=18; P>0.05). Finally, one embryo showed a single deletion for GGTA1 and another showed a double deletion in GGTA1 and VWF genes, out of seven embryos from the CRISPR/Lipo+ group. No gene deletion was confirmed in any of the embryos from the CRISPR/Lipo− group. These results are preliminary data (more experiments are currently being done) suggesting that microinjection of CRISPR/Cas9 with lipofectamine could be used as an alternative delivery system, since it seems to have no impact on porcine embryo development.
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Moore, Anne C., Wing-pui Kong, Bimal K. Chakrabarti, and Gary J. Nabel. "Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice." Journal of Virology 76, no. 1 (January 1, 2002): 243–50. http://dx.doi.org/10.1128/jvi.76.1.243-250.2002.
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ABSTRACT The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-γ) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-γ and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-γ and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.
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Puri, Nakul, Carol Jenner, Mark Bennett, Ruth Stewart, John Mansfield, Nigel Lyons, and John Taylor. "Expression of avrPphB, an Avirulence Gene from Pseudomonas syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean." Molecular Plant-Microbe Interactions® 10, no. 2 (March 1997): 247–56. http://dx.doi.org/10.1094/mpmi.1997.10.2.247.
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Protein production encoded by the avirulence gene avrPphB from Pseudomonas syringae pv. phaseolicola was examined. Incorporation of [35S]-labeled methionine into the AvrPphB protein indicated processing of the full-length peptide in Escherichia coli to give a major 28-kDa product. The 28-kDa native peptide was isolated from E. coli following over-expression of avrPphB and found not to elicit the hypersensitive response (HR) after infiltration into bean leaves. Antiserum raised to the 28-kDa peptide allowed expression of avrPphB and processing of AvrPphB protein to be examined in P. syringae pv. phaseolicola; immunoreactive peptides of both 35 and 28-kDa were detected in races 3 and 4 (which contain avrPphB) only after induction in minimal medium + 10 mM sucrose. Antiserum raised to a synthetic peptide, derived from the sequence of the 62 amino acids found to be cleaved from the full-length AvrPphB protein, revealed the accumulation of peptides corresponding to the smaller cleavage products, in both E. coli and P. syringae pv. phaseolicola. Biochemical localization experiments showed that all AvrPphB peptides were cytoplasmic in P. syringae pv. phaseolicola. No AvrPphB peptides were produced in a hrpL mutant unless expression of the gene was directed by a strong vector promoter; induction kinetics similar to wild type were observed in a hrpY - strain, although it also failed to cause a confluent HR. Growth of P. syringae pv. phaseolicola under inducing conditions removed the requirement for rifampicin-sensitive mRNA synthesis by bacteria to allow HR development (the induction time) in bean and lettuce leaves. Constitutive expression of hrpL reduced but did not remove the induction time. Expression of the hrp gene cluster of P. syringae pv. phaseolicola from plasmid pPPY430 in E. coli enabled phenotypic expression of avrPphE (also carried by pPPY430) and avrPphB (if over-expressed from pPPY3031). Despite constitutive expression of the hrp and avr genes in E. coli, a protein synthesis dependent induction time was still required for development of the HR in bean genotypes with matching resistance genes. The significance of processing for the function of AvrPphB peptides and the delivery of elicitors of the HR are discussed.
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Huang, Yan, George Hajishengallis, and Suzanne M. Michalek. "Construction and Characterization of aSalmonella enterica Serovar Typhimurium Clone Expressing a Salivary Adhesin of Streptococcus mutans under Control of the Anaerobically Inducible nirB Promoter." Infection and Immunity 68, no. 3 (March 1, 2000): 1549–56. http://dx.doi.org/10.1128/iai.68.3.1549-1556.2000.
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ABSTRACT Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to the gut-associated lymphoid tissues. One potential problem associated with this vaccine approach is the likelihood of in vivo instability of the plasmid constructs caused by constitutive hyperexpression of the heterologous immunogen. The aim of this study was to generate and characterize an expression system encoding the saliva-binding region (SBR) of Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2/B subunits (CTA2/B), under the control of the inducible nirB promoter. This promoter is activated in an anaerobic environment and within macrophages, which are the primary antigen-presenting cells involved in phagocytosis and processing of Salmonella. The gene encoding the chimeric SBR-CTA2/B was amplified by PCR using primers containing appropriate restriction sites for subcloning into pTETnirB, which contains the nirB promoter. The resulting plasmid was introduced into serovar Typhimurium by electroporation. Production of the SBR-CTA2/B chimeric protein under anaerobic conditions was verified by enzyme-linked immunosorbent assay of whole-cell lysates on plates coated with GM1 ganglioside and developed with antibodies to SBR. Similar procedures were followed for cloning the gene encoding SBR in serovar Typhimurium undernirB control. Anaerobic expression of SBR was confirmed by Western blotting of whole-cell lysates probed with anti-SBR antibodies. The resulting serovar Typhimurium strains were administered by either the oral or the intranasal route to mice, and colonization was assessed by microbiologic analysis of dissociated spleens, Peyer's patches (PP), and nasal tissues. High numbers of the recombinant strains persisted in PP and spleen for at least 21 days following oral challenge. A single intranasal administration of theSalmonella clones to mice also resulted in the colonization of the nasal tissues by the recombinant bacteria. Salmonellae were recovered from nasal lymphoid tissues, superficial lymph nodes, internal jugular lymph nodes, PP, and spleens of mice for at least 21 days after challenge. This study provides quantitative evidence for colonization by Salmonella strains expressing a recombinant protein under the control of the inducible nirB promoter in PP or nasal tissues following a single oral or nasal administration of the bacteria, respectively.
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Jorasa, Paradon, Phenjun Mekvichitsaeng, and Yaowaluck Maprang Roshorm. "Generation of recombinant Bacillus subtilis expressing Porcine Epidemic Diarrhea Virus (PEDV) S1 protein in vegetative cell." International Journal of Engineering & Technology 7, no. 2.10 (April 2, 2018): 50. http://dx.doi.org/10.14419/ijet.v7i2.10.10953.
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Porcine epidemic diarrhea virus (PEDV) is a mucosal (gut surface) pathogen that causes severe diarrhea in piglets; thus, a vaccine capable of inducing gut-mucosal immune response is crucial for controlling PEDV infection. Bacillus subtilis has been considered a choice for vaccine delivery to the gut mucosa. In this study, we aimed to generate recombinant B. subtilis that can produce PEDV S1 protein in vegetative cell. Two promoters, PrrnO and PgsiB-PsecA, were selected for an early and high yield expression of PEDV S1 gene in B. subtilis vegetative cell and germinating spore. Promoters, PrrnO and PgsiB-PsecA, were linked to the 5’ end of the fusion gene pgsA-PEDVS1 and the fusion genes were then inserted into plasmid pDG1662. Recombinant B. subtilis strains were generated by integrating the fusion genes into B. subtilis 168 chromosome via double crossover homologous recombination. PCR amplification and amylase activity assay confirmed integration of the fusion genes into B. subtilis chromosome at amyE locus. Expression of the pgsA-PEDVS1 in B. subtilis vegetative cells germinating from spores was then studied at 2, 4, 8 and 16 hours of culture. Tested by western blot analysis, although only cleaved products of PgsA-PEDVS1 protein were observed, expression levels of pgsA-PEDVS1 under the control of both promoters were comparable at all time points. Importantly, PgsA-PEDVS1 protein could be detected as early as 2 hours after spore inoculation in LB medium. This study suggests that both PgsiB-PsecA and PrrnO promoters can be used for efficient production of PEDV S1 in germinating spore and vegetative cell and may be applicable for expression of other heterologous genes in B. subtilis vegetative cell.
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Kremer, Laurent, Loïc Dupré, Gilles Riveau, André Capron, and Camille Locht. "Systemic and Mucosal Immune Responses after Intranasal Administration of Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Expressing Glutathione S-Transferase from Schistosoma haematobium." Infection and Immunity 66, no. 12 (December 1, 1998): 5669–76. http://dx.doi.org/10.1128/iai.66.12.5669-5676.1998.
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ABSTRACT A major goal of current vaccine development is the induction of strong immune responses against protective antigens delivered by mucosal routes. One of the most promising approaches in that respect relies on the use of live recombinant vaccine carriers. In this study,Mycobacterium bovis BCG was engineered to produce an intracellular glutathione S-transferase fromSchistosoma haematobium (Sh28GST). The gene encoding Sh28GST was placed under the control of the mycobacterialhsp60 promoter on a replicative shuttle plasmid containing a mercury resistance operon as the only selectable marker. The recombinant Sh28GST produced in BCG bound glutathione and expressed enzymatic activity, indicating that its active site was properly folded. Both intraperitoneal and intranasal immunizations of BALB/c mice with the recombinant BCG resulted in strong anti-Sh28GST antibody responses, which were enhanced by a boost. Mice immunized intranasally produced a mixed response with the production of Sh28GST-specific immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA in the serum. In addition, high levels of anti-Sh28GST IgA were also found in the bronchoalveolar lavage fluids, demonstrating that intranasal delivery of the recombinant BCG was able to induce long-lasting secretory and systemic immune responses to antigens expressed intracellularly. Surprisingly, intranasal immunization with the BCG producing the Sh28GST induced a much stronger specific humoral response than intranasal immunization with BCG producing the glutathioneS-transferase from Schistosoma mansoni, although the two antigens have over 90% identity. This difference was not observed after intraperitoneal administration.
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Kostyrko, Vitaly A., Yulia V. Bertsova, Marina V. Serebryakova, Alexander A. Baykov, and Alexander V. Bogachev. "NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na+-Translocating NADH:Quinone Oxidoreductase." Journal of Bacteriology 198, no. 4 (December 7, 2015): 655–63. http://dx.doi.org/10.1128/jb.00757-15.
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ABSTRACTNa+-translocating NADH:quinone oxidoreductase (Na+-NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na+translocation across the membrane. Na+-NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by thenqr-associatedapbEgene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamednqrM) that usually follows theapbEgene and is present only in Na+-NQR-containing bacteria. Expression of theVibrio harveyinqroperon alone or with the associatedapbEgene inEscherichia coli, which lacks its own Na+-NQR, resulted in an enzyme incapable of Na+-dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na+-NQR was restored when these genes were coexpressed with theV. harveyinqrMgene. Furthermore,nqrMlesions inKlebsiella pneumoniaeandV. harveyiprevented production of functional Na+-NQR, which could be recovered by annqrM-containing plasmid. The Na+-NQR complex isolated from thenqrM-deficient strain ofV. harveyilacks several subunits, indicating thatnqrMis necessary for Na+-NQR assembly. The protein product of thenqrMgene, NqrM, contains a single putative transmembrane α-helix and four conserved Cys residues. Mutating one of these residues (Cys33 inV. harveyiNqrM) to Ser completely prevented Na+-NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na+-NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys)4[Fe] center between subunits NqrD and NqrE.IMPORTANCENa+-translocating NADH:quinone oxidoreductase complex (Na+-NQR) is a unique primary Na+pump believed to enhance the vitality of many bacteria, including important pathogens such asVibrio cholerae,Vibrio parahaemolyticus,Haemophilus influenzae,Neisseria gonorrhoeae,Pasteurella multocida,Porphyromonas gingivalis,Enterobacter aerogenes, andYersinia pestis. Production of Na+-NQR in bacteria requires Na+-NQR-specific maturation factors. We earlier identified one such factor (ApbE) that covalently attaches flavin residues to Na+-NQR. Here we identify the other protein factor, designated NqrM, and show that NqrM and ApbE suffice to produce functional Na+-NQR from theVibrio harveyinqroperon. NqrM may be involved in Fe delivery to a unique Cys4[Fe] center during Na+-NQR assembly. Besides highlighting Na+-NQR biogenesis, these findings suggest a novel drug target to combat Na+-NQR-containing bacteria.
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Shirley, Shawna A., Cathryn G. Lundberg, and Richard Heller. "Electrotransfer of IL-15/IL-15Rα Complex for the Treatment of Established Melanoma." Cancers 12, no. 10 (October 21, 2020): 3072. http://dx.doi.org/10.3390/cancers12103072.
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Gene electrotransfer (GET) is a safe, reliable, and effective method of delivering plasmid DNA (pDNA) to solid tumors. GET has been previously used to deliver interleukin-15 (IL-15) to mouse melanoma, resulting in long-term tumor regression and the survival of a percentage of treated animals after challenge. To enhance this effect, we evaluated modulating the expression levels of IL-15 and co-expressing its receptor, IL-15Rα. GET was used to deliver plasmids encoding IL-15 and IL-15Rα to established B16.F10 tumors on days 0, 4, and 7. Two delivery protocols that yielded different expression profiles were utilized. Mice that were tumor-free for 50 days were then challenged with B16.F10 cells on the opposite flank and monitored for an additional 50 days. The amount of IL-15 expressed and the presence or absence of IL-15Rα in the treated tumors did not significantly affect the tumor regression and long-term survival. Upon challenge, however, low levels of IL-15 were more protective and resulted in a greater production of anti-tumor cytokines such as IFN-γ and MIP-1β and a greater amount of CD11b+ and CD3e+ cells infiltrating tumors. While mice with high levels of IL-15 showed CD11b+ and CD3e+ cell infiltrate, there was a substantial presence of NK cells that was absent in other treated groups. We can conclude that the level of IL-15 expressed in tumors after GET is an important determinant of the therapeutic outcome, a finding that will help us finetune this type of therapy.
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Epperly, Michael W., Yunyun Niu, Hongmei Shen, and Joel S. Greenberger. "Pretreatment of the Esophagus with Manganese Superoxide Dismutase Plasmid/Liposome Complex (MnSOD-PL) before Irradiation Results in Increased Migration and Proliferation of Marrow-Derived Stem Cell Progenitors in the Esophageal Squamous Epithelium." Blood 108, no. 11 (November 16, 2006): 5478. http://dx.doi.org/10.1182/blood.v108.11.5478.5478.
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Abstract Irradiation of the mouse esophagus increases production of superoxide, nitric oxide, peroxynitrite, and other reactive oxygen species (ROS) for weeks after exposure. ROS production in the esophageal microenvironment may be toxic to cells migrating in to repair irradiation damage. MnSOD-PL gene therapy decreases ROS production. We tested whether such treatment increased the engraftment of circulating marrow origin cells involved in repair of esophageal irradiation damage. C57BL/6NHsd female mice were treated with intraesophageal MnSOD-PL and irradiated 24 hr later along with control mice to 29 Gy to the thoracic cavity. Subgroups of mice were sacrificed at days 1, 2, 3, 4, or 5 days after irradiation, esophagus removed, frozen in OCT and sectioned. Sections were stained and apoptosis quantitated. Low level apoptosis was detected for the first 3 days with significant apoptosis on day 5. Mice pretreated with MnSOD-PL had reduced apoptosis (3 ± 1%) compared to control irradiated mice (20 ± 7%) (p= 0.010). Female mice in each 29 Gy esophagus irradiated group were injected I.V. 5 days later with 1 × 105 bone marrow cells from male ROSA mice. Subgroups received MnSOD-PL (100 ug plasmid DNA in 100 ul) 24 hr before irradiation. ROSA donor cells were differentiated from recipient C57BL/6NHsd cells by Y probe and LacZ positive staining and resistance to G418 follows explants in vitro. At sacrifice 14 days after irradiation, esophagus was removed and sorted into side population (SP) and non-side population cells (NSP). Mice that received MnSOD-PL before irradiation, had 42.3 ± 5.3% of NSP cells LacZ+ compared to 10.9 ± 1% in the irradiation only group (p < 0.0001). SP cells from MnSOD-PL treated mice demonstrated 26.5 ± 4.8% LacZ+ cells compared to 9.3 ± 2.0% in the irradiation control group (p = 0.0021). Serial transfer of donor ROSA marrow origin esophageal “stem cell candidates” was carried out by I.V. transfer of 1st generation SP and NSP harvests to 29 Gy thoracic irradiated female recipients. Second generation recipients pretreated with intraesophageal MnSOD-PL who received ROSA male SP cells isolated from first generation mice (also pretreated with MnSOD-PL) demonstrated the highest number of LacZ+ cells (4.7 ± 1.6%). In contrast, no LacZ+, ROSA cells were detected in second generation irradiation control recipients. Therefore, intraesophageal delivery of MnSOD-PL increases stable migration of bone marrow derived progenitors of esophageal squamous epithelium and improves the stability of serially transferred marrow origin, esophageal stem cell candidates.
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Dinh, Long V., Eli Fine, Afshin Ameri, Bernadette W. Luu, Satish Kumar, Vincent P. Diego, Joanne E. Curran, et al. "Specific Correction of the Intron-22 Inverted Factor VIII Gene in Autologous Blood Outgrowth Endothelial Cells from Patients with Severe Hemophilia A." Blood 136, Supplement 1 (November 5, 2020): 30–31. http://dx.doi.org/10.1182/blood-2020-142679.
Full textAbstract:
A heterogeneous collection of >1500 distinct causative factor (F) VIII gene (F8) mutations have been identified thus far in unrelated severe Hemophilia A (HA) patients, who have less than 1% of normal FVIII activity in their plasmas and experience recurrent bleeding that often results in crippling arthropathies and can be life threatening. Curative gene therapies are being pursued intensely as the current standard of care, which involves 2-3 infusions/week of therapeutic FVIII (tFVIII) proteins throughout a patient's life, is extremely expensive and very demanding. Moreover, ~25% of patients with severe HA (PSHA) develop anti-tFVIII antibodies that neutralize the efficacy of their tFVIII proteins. Despite remarkable progress in clinical trials of various adeno-associated viruses (AAVs) as vectors for in vivo delivery of therapeutic F8 genes, it is not clear how widespread viral-mediated gene replacement therapy (GRT) will become due to current limitations that include the: (1) presence of existing immunity to the AAV capsid protein (CP) in ~30-70% of PSHA for whom GRT is contraindicated; (2) immunity to AAV-CP induced in all PSHA during the initial GRT that precludes subsequent dosing; (3) use of heterologous promoters which drive F8 expression in non-physiologic cells that may increase the encoded tFVIII protein's immunogenicity; and 4) episomal location of AAV-genome replication, which, together with "(2)" and "(3)", precludes GRT in children. These important unmet needs require new gene-based therapeutic strategies for HA. Our goal was to develop a virus-free, ex vivo personalized gene repair therapy that minimally manipulates the mutant F8 in autologous patient-derived blood outgrowth endothelial cells (BOECs)-the physiologically relevant cell type for FVIII production in vivo-followed by their expansion and reinfusion into the same individual patient. We chose to focus initially on the intron (I) 22 inversion (I22I) mutation initially as it is causative in >40% of all PSHA. CRISPR/Cas9 guide RNAs were designed to target the 3' end of F8 exon (E) 22. For initial experiments in K562 cells, a donor plasmid containing a restriction enzyme site was nucleofected with the CRISPR system encoding plasmid, which triggered successful homology-directed repair (HDR) at the target site (efficiency of 18.2%, n=2). Subsequently, a cDNA-based therapeutic donor plasmid was constructed containing all F8 coding sequences in E23-E26 followed by a bGH polyA signal. After appropriate informed consent was obtained, BOECs were cultured from the blood of 3 severe pediatric HA patients with the I22I (ages 6 years, 7 years, and 13 years). After nucleofecting the BOECs with the F8-specific CRISPR and HDR constructs, site-specific knock-in of the cDNA at the 3' end of E22 was confirmed via PCR (n=3). Direct Sanger sequencing of the resultant amplicons from the repaired I22-inverted F8 locus in treated BOECs from one representative patient confirmed complete and seamless knock-in of the therapeutic cDNA at the endogenous site of the mutant F8. Current efforts are to isolate clonal populations of the repaired BOECs and characterize their ability to secrete active FVIII in vitro. Similar experiments are underway using canine BOECs in the canine model of HA. The use of clonal populations of gene corrected autologous BOECs as the infused therapy allows whole genome sequencing analysis to be performed to confirm that no off-target cutting or integration occurred in the therapeutic cell preparation prior to infusion into the recipient patient, further strengthening the safety profile of this proposed autologous cell therapy. Overall, these current results lay promising proof-of-concept data for a potential new curative therapeutic alternative approach for HA which should overcome drawbacks of the current generations of AAV-based treatments. Disclosures Dinh: Haplogenics Corporation: Current Employment. Luu:Haplogenics Corporation: Current Employment. Mead:CSL Behring: Current Employment. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Powell:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees. Howard:Haplogenics Corporation: Membership on an entity's Board of Directors or advisory committees.
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Barnett, Burton Earle, David L. Hermanson, Jenessa Barbara Smith, Xinxin Wang, Yening Tan, Christopher E. Martin, Eric M. Osertag, and Devon J. Shedlock. "piggyBacTM-Produced CAR-T Cells Exhibit Stem-Cell Memory Phenotype." Blood 128, no. 22 (December 2, 2016): 2167. http://dx.doi.org/10.1182/blood.v128.22.2167.2167.
Full textAbstract:
Abstract Immunotherapy using chimeric-antigen receptor (CAR)-T cells is emerging as an exciting therapeutic approach for cancer therapies. Autologous CAR-modified T cells targeting a tumor-associated antigen (Ag) can result in robust tumor killing, in some cases resulting in complete remission of CD19+ hematological malignancies. Unlike traditional biologics and chemotherapeutics, CAR-T cells possess the capacity to rapidly reproduce upon Ag recognition, thereby potentially obviating the need for repeat treatments. To achieve this, CAR-T cells must not only drive tumor destruction initially, but must also persist in the patient as a stable population of viable memory T cells to prevent potential cancer relapses. Thus, intensive efforts have been focused on the development of CAR molecules that do not cause T cell exhaustion through Ag-independent (tonic) signaling, as well as of a CAR-T product containing early memory cells, especially stem cell memory (TSCM). It is hypothesized that a stem cell-like CAR-T would exhibit the greatest capacity for self-renewal and multipotent capacity to derive central memory (TCM), effector memory (TEM) and effector T cells (TE), thereby producing better tumor eradication and long-term CAR-T engraftment. We developed a novel Centyrin-based CAR, referred to as a CARTyrin, that is specific for human B cell maturation antigen (BCMA). Centyrins are alternative scaffold molecules based on human consensus tenascin FN3 domain, are smaller than scFv molecules, and can be selected for monomeric properties that favor stability and decrease the likelihood of tonic signaling in CAR molecules. We produced a plasmid DNA transposon encoding the CARTyrin that was flanked by two cis-regulatory insulator elements to help stabilize CARTyrin expression by blocking improper gene activation or silencing. The piggyBac™ (PB) Transposon System was used for stable integration of anti-BCMA CARTyrin into resting pan T cells, whereby the transposon was co-delivered along with an mRNA transposase enzyme, called Super piggyBac™ (SPB), in a single electroporation reaction. Delivery of piggyBac™ transposon into untouched, resting primary human pan T cells resulted in 20-30% of cells with stable integration and expression of PB-delivered genes. Surprisingly, we observed that a majority of these cells were positive for expression of CD62L and CD45RA, markers commonly associated with TSCM cells. To see if this phenotype was retained upon CAR-T cell stimulation and expansion, we activated the cells via stimulation of CD3 and CD28, and later show that > 60% of CARTyrin+ T cells exhibited a stem-cell memory phenotype. Furthermore, these cells were fully capable of expressing potent anti-tumor effector function. To determine whether or not the PB system directly contributed to enhancing the expression of stem-like markers, we compared the phenotype of CAR-T cells generated either by PB transposition or lentiviral (LV) transduction. To do this, we constructed a new vector by subcloning the CARTyrin transgene into a common LV construct for production of virus. Following introduction of the CARTyrin to untouched resting T cells either by PB-transposition or LV-transduction, we expanded the CARTyrin+ cells and then allowed them to return to a resting state. A variety of phenotypic and functional characteristics were measured including kinetic analysis of memory and exhaustion-associated markers, secondary proliferation in response to homeostatic cytokine or tumor-associated Ag, cytokine production, and lytic capability in response to BCMA+ tumor cells. Unlike the PB-transposed CARTyrin+ T cells, we found that the LV-transduced CARTyrin+ T cells did not exhibit an augmented memory phenotype. In addition, PB-transposed cells exhibited a comparable or greater capability for secondary proliferation and killing of BCMA+ tumor cells. Together, these data demonstrate that CAR-T cells produced by PB transposition are predominantly TSCM cells, a highly desirable product phenotype in the CAR-T field. Furthermore, these CARTyrin+ T cells exhibit strong anti-tumor activity and may give rise to cells that persist longer in vivo due to the use of a Centyrin-based CAR, which may be less prone to tonic signaling and functional exhaustion. Disclosures Barnett: Poseida Therapeutics: Employment. Hermanson:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.