Genetic Vaccines and Therapy最新文献

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 - 10(9) 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.

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 beta-thalassemic mice.

Methods: Muscular electrotransfer of 1 microg, 1.5 microg, 2 microg, 4 microg or 6 microg of Epo-plasmid was performed in beta-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 microg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in beta-thalassemic mice for more than 9 months.

Conclusion: This strategy led to efficient, long-lasting and non-toxic treatment of beta-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.

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 x 10(11) or 5 x 10(11) 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.

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.

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.

Bovine herpesvirus 4 (BoHV-4) is a gamma-herpesvirus with no clear disease association. A recombinant BoHV-4 (BoHV-4EGFP Delta TK) expressing Green Fluorescent Protein (EGFP), was successfully used to infect F98 rat glioma cells. BoHV-4EGFP Delta TK 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.

It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern.

Tattooing is one of a number of DNA delivery methods which results in an efficient expression of an introduced gene in the epidermal and dermal layers of the skin. The tattoo procedure causes many minor mechanical injuries followed by hemorrhage, necrosis, inflammation and regeneration of the skin and thus non-specifically stimulates the immune system. DNA vaccines delivered by tattooing have been shown to induce higher specific humoral and cellular immune responses than intramuscularly injected DNA. In this study, we focused on the comparison of DNA immunization protocols using different routes of administrations of DNA (intradermal tattoo versus intramuscular injection) and molecular adjuvants (cardiotoxin pre-treatment or GM-CSF DNA co-delivery). For this comparison we used the major capsid protein L1 of human papillomavirus type 16 as a model antigen. L1-specific immune responses were detected after three and four immunizations with 50 microg plasmid DNA. Cardiotoxin pretreatment or GM-CSF DNA co-delivery substantially enhanced the efficacy of DNA vaccine delivered intramuscularly by needle injection but had virtually no effect on the intradermal tattoo vaccination. The promoting effect of both adjuvants was more pronounced after three rather than four immunizations. However, three DNA tattoo immunizations without any adjuvant induced significantly higher L1-specific humoral immune responses than three or even four intramuscular DNA injections supported by molecular adjuvants. Tattooing also elicited significantly higher L1-specific cellular immune responses than intramuscularly delivered DNA in combination with adjuvants. In addition, the lymphocytes of mice treated with the tattoo device proliferated more strongly after mitogen stimulation suggesting the presence of inflammatory responses after tattooing. The tattoo delivery of DNA is a cost-effective method that may be used in laboratory conditions when more rapid and more robust immune responses are required.

Background: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses.

Methods: To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity.

Results: It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes.

Conclusion: Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

Background: Early prostate adenocarcinoma can be diagnosed through seric prostate-specific antigen (PSA) screenings. However, a fraction of patients progress to an incurable metastatic disease. Therefore, novel therapies for treating these patients are extremely desirable. Therapeutic vaccines based on Dendritic Cells (DCs) carrying tumor antigens have emerged as a promising strategy to initiate an immune response against tumor cells. These vaccines can be prepared using different methodologies, such as the application of tumor mRNA described in this work.

Methods: Mature and immature DCs were obtained in vitro by adding specific cytokines to monocyte cell cultures. RNA extracted from prostate tumor lineage (LNCAP) was introduced into these cells by electroporation and co-incubation. Transfection success was measured by immunocytochemistry of the PSA expression level in DCs.

Results: Cell surface markers, including CD14, CD80, CD86, CCR7, CD11c, and CD1a, confirmed mature and immature DC phenotypes. Both cell maturation stages were successfully used for RNA introduction as shown by PSA characterization.

Conclusion: Our data support the use of mature and immature DCs for vaccine preparation with either RNA electroporation or RNA co-incubation. The highest efficiency, however, was observed when RNA was delivered by electroporation into mature DCs. Due to in vitro RNA transcription, this method allows small tumors to be used for DC vaccine preparation; it is therefore a promising approach for the treatment of metastatic prostate cancer.