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Genetic immunization with Hantavirus vaccine combining expression of G2 glycoprotein and fused interleukin-2
In this research, we developed a novel chimeric HTNV-IL-2-G2 DNA vaccine plasmid by genetically linking IL-2 gene to the G2 segment DNA and tested whether it could be a candidate vaccine. Chimeric gene was first expressed in eukaryotic expression system pcDNA3.1 (+). The HTNV-IL-2-G2 expressed a 72 kDa fusion protein in COS-7 cells. Meanwhile, the fusion protein kept the activity of its parental proteins. Furthermore, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response.- The results showed that the chimeric gene could simultaneously evoke specific antibody against G2 glycoprotein and IL-2. And the immunized mice of every group elicited neutralizing antibodies with different titers. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to G2 and IL-2 were significantly higher than that of other groups. Our results suggest that IL-2-based HTNV G2 DNA can induce both humoral and cellular immune response specific for HTNV G2 and can be a candidate DNA vaccine for HTNV infection.
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Relative persistence of AAV serotype 1 vector genomes in dystrophic muscle
The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation.One day old alpha sarcoglycan knockout mice (sgca-/-) were injected with 1 × 1011 vector genomes of rAAV2/1-tMCK-sgca in one hindlimb and the same dose of rAAV2/1-tMCK-LacZ in the contra lateral hindlimb. Newborn mice are tolerant of the foreign transgene allowing for long-term expression of both the marker and the therapeutic gene in the null background. At 2 time-points following vector administration, hindlimb muscles were harvested and analyzed for LacZ or sarcoglycan expression. Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene. We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer. In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss. Mitigating the membrane instability inherent in dystrophic muscle was able to prolong the life of individual myofibers.
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Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice
The AAV9 capsid displays a high natural affinity for the heart following a single intravenous (IV) administration in both newborn and adult mice. It also results in substantial albeit relatively lower expression levels in many other tissues. To increase the overall safety of this gene delivery method we sought to identify which one of a group of promoters is able to confer the highest level of cardiac specific expression and concurrently, which is able to provide a broad biodistribution of expression across both cardiac and skeletal muscle. The in vivo behavior of five different promoters was compared: CMV, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) and cardiac troponin C (cTnC). Following IV administration to newborn mice, LacZ expression was measured by enzyme activity assays. Results showed that rAAV2/9-mediated gene delivery using the α-MHC promoter is effective for focal transgene expression in the heart and the Des promoter is highly suitable for achieving gene expression in cardiac and skeletal muscle following systemic vector administration. Importantly, these promoters provide an added layer of control over transgene activity following systemic gene delivery.
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Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line
Background:
For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants.
Methods:
To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls.
Results:
Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed.
Conclusion:
Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors.
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Quantitative real-time PCR study on persistence of pDNA vaccine pVax-Hsp60 TM814 in beef muscles
Background:
Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.
Methods:
A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 109 copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.
Results:
Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.
Conclusion:
Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.
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Careful adjustment of Epo non-viral gene therapy for β-thalassemic anaemia treatment
Background:
In situ production of a secreted therapeutic protein is one of the major gene therapy applications. Nevertheless, the plasmatic secretion peak of transgenic protein may be deleterious in many gene therapy applications including Epo gene therapy. Epo gene transfer appears to be a promising alternative to recombinant Epo therapy for severe anaemia treatment despite polycythemia was reached in many previous studies. Therefore, an accurate level of transgene expression is required for Epo application safety. The aim of this study was to adapt posology and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak and maintain treatment efficiency. The therapeutic potential of repeated muscular electrotransfer of light Epo-plasmid doses was evaluated for anaemia treatment in β-thalassemic mice.
Methods:
Muscular electrotransfer of 1 μg, 1.5 μg, 2 μg 4 μg or 6 μg of Epo-plasmid was performed in β-thalassemic mice. Electrotransfer was repeated first after 3.5 or 5 weeks first as a initiating dose and then according to hematocrit evolution.
Results:
Muscular electrotransfer of the 1.5 μg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in β-thalassemic mice for more than 9 months.
Conclusion:
This strategy led to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse anaemia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation amplitude. This repeat delivery protocol of light doses of therapeutic gene could be applied to a wide variety of candidate genes as it leads to therapeutic effect reiterations and increases safety by allowing careful therapeutic adjustments.
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Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats
Background:
Vectors based on adeno-associated virus-8 (AAV8) have shown efficiency and efficacy for liver-directed gene therapy protocols following intravascular injection, particularly in relation to haemophilia gene therapy. AAV8 has also been proposed for gene therapy targeted at skeletal and cardiac muscle, again via intravascular injection. It is important to assess vector targeting at the level of virion accumulation and transgene expression in multiple species to ascertain potential issues relating to species variation in infectivity profiles.
Methods:
We used AAV8 vectors expressing human factor IX (FIX) from the liver-specific LP-1 promoter and administered this virus via the intravascular route of injection into 12 week old Wistar Kyoto rats. We assessed FIX levels in serum by ELISA and transgene expression at sacrifice by immunohistochemistry using anti-FIX antibodies. Vector DNA levels in organs we determined by real time PCR.
Results:
Administration of 1 × 1011 or 5 × 1011 scAAV8-LP1-hFIX vector particles/rat resulted in efficient production of physiological hFIX levels, respectively in blood assessed 4 weeks post-injection. This was maintained for the 4 month duration of the study. At 4 months we observed liver persistence of vector with minimal non-hepatic distribution.
Conclusion:
Our results demonstrate that AAV8 is a robust vector for delivering therapeutic genes into rat liver following intravascular injection.
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Clostridial spores as live 'Trojan horse' vectors for cancer gene therapy: comparison with viral delivery systems
Solid tumours account for 90% of all cancers. Gene therapy represents a potential new modality for their treatment. Up to now, several approaches have been developed, but the most efficient ones are the viral vector based gene therapy systems. However, viral vectors suffer from several deficiencies: firstly most vectors currently in use require intratumoural injection to elicit an effect. This is far from ideal as many tumours are inaccessible and many may have already spread to other parts of the body, making them difficult to locate and inject gene therapy vectors into. Second, because of cell heterogeneity within a given cancer, the vectors do not efficiently enter and kill every cancer cell. Third, hypoxia, a prevalent characteristic feature of most solid tumours, reduces the ability of the viral vectors to function and decreases viral gene expression and production. Consequently, a proportion of the tumour is left unaffected, from which tumour regrowth occurs. Thus, cancer gene therapy has yet to realise its full potential.The facultative or obligate anaerobic bacteria have been shown to selectively colonise and regerminate in solid tumours when delivered systemically. Among them, the clostridial spores were easy to produce, stable to store and safe to use as well as having extensive oncolytic ability. However, research in animals and humans has shown that oncolysis was almost always interrupted sharply at the outer rim of the viable tumour tissue where the blood supply was sufficient. These clostridial spores, though, could serve as "Trojan horse" for cancer gene therapy. Indeed, various spores harbouring genes for cancerstatic factors, prodrug enzymes, or proteins or cytokines had endowed with additional tumour-killing capability. Furthermore, combination of these "Trojan horses" with conventional chemotherapy or radiation therapies often significantly perform better, resulting in the "cure" of solid tumours in a high percentage of animals.It is, thus, not too difficult to predict the potential outcomes for the use of clostridial spores as "Trojan horse" vectors for oncolytic therapy when compared with viral vector-mediated cancer therapy for it be replication-deficient or competent. However, to move the "Trojan horse" to a clinic, though, additional requirements need to be satisfied (i) target tumours only and not anywhere else, and (ii) be able to completely kill primary tumours as well as metastases. Current technologies are in place to achieve these goals.
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Prevention of airway inflammation with topical cream containing imiquimod and small interfering RNA for natriuretic peptide receptor
Background:
Asthma is a complex disease, characterized by reversible airway obstruction, hyperresponsiveness and chronic inflammation. Principle pharmacologic treatments for asthma include bronchodilating beta2-agonists and anti-inflammatory glucocorticosteroids; but these agents do not target the main cause of the disease, the generation of pathogenic Th2 cells. We previously reported reduction in allergic inflammation in mice deficient in the ANP receptor NPRA. Here we determined whether siRNA for natriuretic peptide receptor A (siNPRA) protected against asthma when administered transdermally.
Methods:
Imiquimod cream mixed with chitosan nanoparticles containing either siRNA green indicator (siGLO) or siNPRA was applied to the skin of mice. Delivery of siGLO was confirmed by fluorescence microscopy. The anti-inflammatory activity of transdermal siNPRA was tested in OVA-sensitized mice by measuring airway hyperresponsiveness, eosinophilia, lung histopathology and pro-inflammatory cytokines.
Results:
SiGLO appearing in the lung proved the feasibility of transdermal delivery. In a mouse asthma model, BALB/c mice treated with imiquimod cream containing siNPRA chitosan nanoparticles showed significantly reduced airway hyperresponsiveness, eosinophilia, lung histopathology and pro-inflammatory cytokines IL-4 and IL-5 in lung homogenates compared to controls.
Conclusion:
These results demonstrate that topical cream containing imiquimod and siNPRA nanoparticles exerts an anti-inflammatory effect and may provide a new and simple therapy for asthma.
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Transduction of the rat brain by Bovine Herpesvirus 4
Bovine herpesvirus 4 (BoHV-4) is a gamma-herpesvirus with no clear disease association. A recombinant BoHV-4 (BoHV-4EGFPΔTK) expressing Green Fluorescent Protein (EGFP), was successfully used to infect F98 rat glioma cells. BoHV-4EGFPΔTK was injected into the lateral ventricle of the rat brain. Histology and immunohistochemistry showed that ependymal and rostral migratory stream cells were transduced while neurons were not. Clinical scores, evaluated for 90 days, indicated that the virus was non neuropathogenic, suggesting this virus is a suitable vector for brain tumor gene therapy.
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