http://www.gvt-journal.com The latest research articles published by Genetic Vaccines and Therapy 2009-06-21T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ Background: Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2 (Flk-1/KDR), play a key role in tumor angiogenesis. Blocking the VEGF-Flk-1 pathway may inhibit tumor growth. Here, we used human VEGFR-2 as a model antigen to explore the feasibility of immunotherapy with a plasmid DNA vaccine based on a xenogeneic homologue of this receptor. Methods: The protective effects and therapeutic anti-tumor immunity mediated by the DNA vaccine were investigated in mouse models. Anti-angiogenesis effects were detected by immunohistochemical staining and the alginate-encapsulate tumor cell assay. The mechanism of action of the DNA vaccine was primarily explored by detection of auto-antibodies and CTL activity. Results: The DNA vaccine elicited a strong, therapeutic anti-tumor immunity through an anti-angiogenesis mechanism in mouse models, mediated by the stimulation of an antigen-specific response against mFlk-1. Conclusions: Our study shows that a DNA vaccine based on a xenogeneic homologue plasmid DNA induced autoimmunity against VEGFR-2, resulting in inhibition of tumor growth. Such vaccines may be clinically relevant for cancer immunotherapy. http://www.gvt-journal.com/content/7/1/10 Ke Xie Rui-Zhen Bai Yang Wu Quan Liu Kang Liu Yu-Quan Wei Genetic Vaccines and Therapy 2009, 7:10 2009-06-21T00:00:00Z doi:10.1186/1479-0556-7-10 Genetic Vaccines and Therapy 1479-0556 7 10 2009-06-21T00:00:00Z PDF In a mouse model for molybdenum cofactor deficiency as an example for an inherited metabolic disease we have determined the dosage of recombinant AAV necessary to rescue the lethal deficiency phenotype. We demonstrated long-term expression of different expression cassettes delivered in a chimeric AAV capsid of serotype 1/2 and compared different routes of application. We then studied the effect of double and triple injections at different time points after birth and found a short neonatal window for non-response of the immune system. Exposition with rAAV capsids within this window allows transgene expression after a second rAAV transduction later. However, exposition within this window does not trigger immunotolerance to the viral capsid, which limits rAAV-mediated refurbishment of the transgene to only one more application outside this permissive window. http://www.gvt-journal.com/content/7/1/9 Rita Hahnewald Waja Wegner Jochen Reiss Genetic Vaccines and Therapy 2009, 7:9 2009-06-18T00:00:00Z doi:10.1186/1479-0556-7-9 Genetic Vaccines and Therapy 1479-0556 7 9 2009-06-18T00:00:00Z XML Background: The use of shRNAs to downregulate the expression of specific genes is now relatively routine in experimentation but still hypothetical for clinical application. A potential therapeutic approach for HIV-1 disease is shRNA mediated downregulation of the HIV-1 co-receptor, CCR5. It is increasingly recognized that siRNAs and shRNAs can have unintended consequences such as cytotoxicities in cells, particularly when used for long term therapeutic purposes. For the clinical use of shRNAs, it is crucial to identify a shRNA that can potently inhibit CCR5 expression without inducing unintended cytotoxicities. Results: Previous shRNAs to CCR5 identified using conventional commercial algorithms showed cytotoxicity when expressed using the highly active U6 pol III promoter in primary human peripheral blood derived mononuclear cells. Expression using the lower activity H1 promoter significantly reduced toxicity, but all shRNAs also reduced RNAi activity. In an effort to identify shRNAs that were both potent and non-cytotoxic, we created a shRNA library representing all potential CCR5 20 to 22-nucleotide shRNA sequences expressed using an H1 promoter and screened this library for downregulation of CCR5. We identified one potent CCR5 shRNA that was also non-cytotoxic when expressed at a low level with the H1 promoter. We characterized this shRNA in regards to its function and structure. This shRNA was unique that the use of commercial and published algorithms to predict effective siRNA sequences did not result in identification of the same shRNA. We found that this shRNA could induce sequence specific reduction of CCR5 at post transcriptional level, consistent with the RNA interference mechanism. Importantly, this shRNA showed no obvious cytotoxicity and was effective at downregulating CCR5 in primary human peripheral blood derived mononuclear cells. Conclusion: We report on the characterization of a rare shRNA with atypical structural features having potent RNAi activity specific to CCR5. These results have implications for the application of RNAi technology for therapeutic purposes. http://www.gvt-journal.com/content/7/1/8 Saki Shimizu Masakazu Kamata Panyamol Kittipongdaja Kevin Chen Sanggu Kim Shen Pang Joshua Boyer F.Xiao-Feng Qin Dong An Irvin Chen Genetic Vaccines and Therapy 2009, 7:8 2009-06-10T00:00:00Z doi:10.1186/1479-0556-7-8 Genetic Vaccines and Therapy 1479-0556 7 8 2009-06-10T00:00:00Z XML Background: Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. Methods: Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1–98 and 1–173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-μg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 μg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 μg of core aa 1–98 in prime and boost, or with 100 μg of pCMVcoreKozak in prime and 20 μg of core aa 1–98 in boost (III). Antibody response, [3H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. Results: Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 ± 0.18, 0.83 ± 0.5, and 13 ± 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-γ and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 103(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-γ secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regimen that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer ≥ 3 × 103. Conclusion: Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regimen that circumvents the negative effects of intracellular core expression. http://www.gvt-journal.com/content/7/1/7 Ekaterina Alekseeva Irina Sominskaya Dace Skrastina Irina Egorova Elizaveta Starodubova Eriks Kushners Marija Mihailova Natalia Petrakova Ruta Bruvere Tatyana Kozlovskaya Maria Isaguliants Paul Pumpens Genetic Vaccines and Therapy 2009, 7:7 2009-06-08T00:00:00Z doi:10.1186/1479-0556-7-7 Genetic Vaccines and Therapy 1479-0556 7 7 2009-06-08T00:00:00Z XML Background: Nucleofection is an emerging technology for delivery of nucleic acids into both the cytoplasm and nucleus of eukaryotic cells with high efficiency. This makes it an ideal technology for gene delivery and siRNA applications. A 96-well format has recently been made available for high-throughput nucleofection, however conditions must be optimized for delivery into each specific cell type. Screening each 96-well plate can be expensive, and descriptions of methods and outcomes to determine the best conditions are lacking in the literature. Here we employ simple methods, including cell counting, microscopy, viability and cytotoxicity assays to describe the minimal experimental methods required to optimize nucleofection conditions for a given cell line. Methods: We comprehensively measured and analyzed the outcomes of the 96-well nucleofection of pmaxGFP plasmids encoding green fluorescent protein (GFP) into the A-549 human lung epithelial cell line. Fluorescent microscopy and a plate reader were used to respectively observe and quantify green fluorescence in both whole and lysed cells. Cell viability was determined by direct counting/permeability assays, and by both absorbance and fluorescence-based plate reader cytotoxicity assays. Finally, an optimal nucleofection condition was used to deliver siRNA and gene specific knock-down was demonstrated. Results: GFP fluorescence among conditions ranged from non-existent to bright, based upon the fluorescent microscopy and plate reader results. Correlation between direct counting of cells and plate-based cytotoxicity assays were from R = .81 to R = .88, depending on the assay. Correlation between the GFP fluorescence of lysed and unlysed cells was high, ranging from R = .91 to R = .97. Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70–95% knock down of the gene over 48 h with 90–97% cell viability. Conclusion: Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549. We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells. Our analysis of outcomes suggests the need to only measure unlysed, whole-cell fluorescence and cell metabolic activity using a plate reader cytotoxicity assay to determine the best conditions for 96-well nucleofection. http://www.gvt-journal.com/content/7/1/6 Christopher Bradburne Kelly Robertson Dzung Thach Genetic Vaccines and Therapy 2009, 7:6 2009-05-11T00:00:00Z doi:10.1186/1479-0556-7-6 Genetic Vaccines and Therapy 1479-0556 7 6 2009-05-11T00:00:00Z XML The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metasasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate reduction in primary subcutaneous tumour growth. Overall, this study demonstrates the potential for Nk4 gene therapy of metastatic tumours, when delivered by AAV or non-viral methods. http://www.gvt-journal.com/content/7/1/5 Alexandra Buhles Sara Collins Jan van Pijkeren Simon Rajendran Michelle Miles Gerald O'Sullivan Deirdre O'Hanlon Mark Tangney Genetic Vaccines and Therapy 2009, 7:5 2009-03-09T00:00:00Z doi:10.1186/1479-0556-7-5 Genetic Vaccines and Therapy 1479-0556 7 5 2009-03-09T00:00:00Z XML Background: The use of food-grade lactococci as bacterial carriers to DNA delivery into epithelial cells is a new strategy to develop live oral DNA vaccine. Our goal was to develop a new plasmid, named pValac, for antigen delivery for use in lactococci. The pValac plasmid was constructed by the fusion of: i) a eukaryotic region, allowing the cloning of an antigen of interest under the control of the pCMV eukaryotic promoter to be expressed by a host cell and ii) a prokaryotic region allowing replication and selection of bacteria. In order to evaluate pValac functionality, the gfp ORF was cloned into pValac (pValac:gfp) and was analysed by transfection in PK15 cells. The applicability of pValac was demonstrated by invasiveness assays of Lactococcus lactis inlA+ strains harbouring pValac:gfp into Caco-2 cells. Results: After transfection with pValac:gfp, we observed GFP expression in PK15 cells. L. lactis inlA+ were able to invade Caco-2 cells and delivered a functional expression cassette (pCMV:gfp) into epithelial cells. Conclusion: We showed the potential of an invasive L. lactis harbouring pValac to DNA delivery and subsequent triggering DNA expression by epithelial cells. Further work will be to examine whether these strains are able to deliver DNA in intestinal cells in vivo. http://www.gvt-journal.com/content/7/1/4 Valeria Guimaraes Sylvia Innocentin Jean-Marc Chatel Francois Lefevre Philippe Langella Vasco Azevedo Anderson Miyoshi Genetic Vaccines and Therapy 2009, 7:4 2009-02-10T00:00:00Z doi:10.1186/1479-0556-7-4 Genetic Vaccines and Therapy 1479-0556 7 4 2009-02-10T00:00:00Z XML Since publication of our article "Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice" Christina A Pacak, Yoshihisa Sakai, Bijoy D Thattaliyath, Cathryn S Mah and Barry J Byrne Genetic Vaccines and Therapy 2008, 6:13, it has come to our attention that the alpha-myosin heavy chain promoter was incorrectly identified as being of human origin. The correct origin of the promoter was from mouse. http://www.gvt-journal.com/content/7/1/3 Christina Pacak Yoshihisa Sakai Bijoy Thattaliyath Cathryn Mah Barry Byrne Genetic Vaccines and Therapy 2009, 7:3 2009-02-04T00:00:00Z doi:10.1186/1479-0556-7-3 Genetic Vaccines and Therapy 1479-0556 7 3 2009-02-04T00:00:00Z XML Background: Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration. Methods: In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine. Results: The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicty T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunityas indicated by depletion of lymphocytes in vivo. Conclusion: Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future. http://www.gvt-journal.com/content/7/1/2 Ming-Derg Lai Meng-Chi Yen Chiu-Mei Lin Cheng-Feng Tu Chun-Chin Wang Pei-Shan Lin Huei-Jiun Huang Chi-Chen Lin Genetic Vaccines and Therapy 2009, 7:2 2009-01-30T00:00:00Z doi:10.1186/1479-0556-7-2 Genetic Vaccines and Therapy 1479-0556 7 2 2009-01-30T00:00:00Z XML Background: The delivery of therapeutic genes to the central nervous system (CNS) using viral vectors represents an appealing strategy for the treatment of nerve injury and disorders of the CNS. Important factors determining CNS targeting include tropism of the viral vectors and retrograde transport of the vector particles. Retrograde transport of equine anemia virus (EIAV)-based lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus RabERA strain from peripheral muscle to spinal motor neurons (MNs) was previously reported. Despite therapeutic effects achieved in mouse models of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the efficiency of this approach needs to be improved for clinical translation. To date there has not been a quantitative assessment of pseudotyped HIV-1-based lentiviral vectors to transduce MNs. Here, we describe quantitative tests to analyze the retrograde transport capacity of HIV-1 vectors pseudotyped with the G glycoprotein derived from Rabies and Rabies-related viruses (Lyssaviruses). Methods: With a view toward optimizing the retrograde transport properties of HIV-1-based lentiviral vectors, we compared the glycoproteins from different enveloped viruses belonging to the Rhabdoviridae family, genus Lyssavirus, and evaluated their ability to transduce specific cell populations and promote retrograde axonal transport. We first tested the transduction performance of these pseudotypes in vitro in SH-SY5Y neuroblastoma cells, NSC-34 neuroblastoma-spinal cord hybrid cells, and primary mixed spinal cord and pure astrocyte cultures. We then analyzed the uptake and retrograde transport of these pseudotyped vectors in vitro, using Campenot chambers. Finally, intraneural injections were performed to evaluate the in vivo retrograde axonal transport of these pseudotypes. Results: Both the in vitro and in vivo studies demonstrated that lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus PV strain possessed the best performance and neuronal tropism among the vectors tested. Conclusion: Our results indicate that HIV-1-based lentiviral vectors pseudotyped with the Rabies PV glycoprotein might provide important vehicles for CNS targeting by peripheral injection in the treatment of motor neuron diseases (MND), pain, and neuropathy. http://www.gvt-journal.com/content/7/1/1 Thais Federici Robert Kutner Xian-Yang Zhang Hitoshi Kuroda Noel Tordo Nicholas Boulis Jakob Reiser Genetic Vaccines and Therapy 2009, 7:1 2009-01-13T00:00:00Z doi:10.1186/1479-0556-7-1 Genetic Vaccines and Therapy 1479-0556 7 1 2009-01-13T00:00:00Z XML