伪狂犬病毒VP22蛋白C-端系列缺失突变体的构建及亚细胞定位分析

以绿荧光蛋白(GFP)为标记,构建了一系列伪狂犬病毒VP22蛋白的C-端缺失突变体与GFP融合表达的真核表达质粒,脂质体介导转染Hela细胞,通过荧光显微镜观察分析各个缺失突变体的亚细胞定位,发现伪狂犬病毒VP22蛋白与核定位有关的结构域在第60个到第90个氨基酸残基之间,第111个到第159个氨基酸残基有可能与形成细胞核内的颗粒有关,与微管蛋白结合有关的结构域可能在第187到第241个氨基酸残基之间。上述研究结果为进一步深入研究伪狂犬病毒VP22蛋白的结构与功能奠定了基础。     

Production of functional recombinant tyrosine hydroxylase by the BPV-1 expression plasmids in the cell cultures

Bovine papilloma virus type-1 (BPV-1)-based expression plasmids TkBPVTH and CGalBPVTH encoding the rat tyrosine hydroxylase (TH) enzyme have been designed for the development of gene therapy for experimental Parkinson's disease. The aim of the present work was to examine the transfection of BPVTH plasmids to express a dopaminergic transgene in the monkey CV1-P fibroblast, rat C6 glioma and human NHA astrocyte cell cultures. The biological function of the transgene was estimated by analyzing the production of recombinant TH mRNA and protein, and the synthesis of L-dopa and dopamine. The highest transfection efficiency was obtained using TkBPVTH plasmids (5 microg). Furthermore, the expression of TkBPVTH plasmids was associated with significant synthesis of TH enzyme and L-dopa in the C6 and NHA cell cultures.     

Poor intercellular transport and absence of enhanced antiproliferative activity after non-viral gene transfer of VP22-P53 or P53-VP22 fusions into p53 null cell lines in vitro or in vivo

BACKGROUND: The herpes simplex virus type 1 (HSV-1) VP22 protein has the property to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus. We have previously shown improved delivery and enhanced therapeutic effect in vitro and in vivo with a P27-VP22 fusion protein. In this report, we were interested in studying the spread and biological activity of VP22 fused to the P53 tumor suppressor. METHODS: Expression of the VP22-P53 and P53-VP22 fusion proteins was shown by Western blot and intercellular spreading was monitored by immunofluorescence on transiently transfected cells. In vitro antiproliferative activity of wild-type (wt) P53 and P53-VP22 was assessed by proliferation assays and transactivating ability was studied by a reporter gene test and a gel-shift assay. Antitumor activity was also tested in vivo by intratumoral injections of naked DNA in a model of subcutaneous tumors implanted in nude mice. RESULTS: Our results show that the C-terminal fusion or the N-terminal P53-VP22 fusion proteins are not able to spread as efficiently as VP22. Moreover, we demonstrate that VP22-P53 does not possess any transactivating ability. P53-VP22 has an antiproliferative activity, but this activity is not superior to the one of P53 alone, in vitro or in vivo. CONCLUSIONS: Our study indicates that a gene transfer strategy using VP22 cannot be considered as a universal system to improve the delivery of any protein.     

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications

BACKGROUND: Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo. METHODS: Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo. RESULTS: Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days. CONCLUSIONS: FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.     

Transfection of bovine fetal fibroblast with polyethylenimine (PEI) nanoparticles: effect of particle size and presence of fetal bovine serum on transgene delivery and cytotoxicity

The development of efficient transfection protocols for livestock cells is crucial for implementation of cell-based transgenic methods to produce genetically modified animals. We synthetized fully deacylated linear 22, 87 and 217 kDa polyethylenimine (PEI) nanoparticles and compared their transfection efficiency and cytotoxicity to commercial branched 25 kDa PEI and linear 58 kDa poly(allylamine) hydrochloride. We studied the effect of PEI size and presence of serum on transfection efficiency on primary cultures of bovine fetal fibroblasts and established cells lines (HEK 293 and Hep G2). We found that transfection efficiency was affected mainly by polymer/pDNA ratio and DNA concentration and in less extent by PEI MW. In bovine fibroblast, preincubation of PEI nanoparticles with fetal bovine serum (FBS) greatly increased percentage of cells expressing the transgene (up to 82%) while significantly decreased the polymer cytotoxic effect. 87 and 217 kDa PEI rendered the highest transfection rates in HEK 293 and Hep G2 cell lines (>50% transfected cells) with minimal cell toxicity. In conclusion, our results indicate that fully deacylated PEI of 87 and 217 kDa are useful DNA vehicles for non-viral transfection of primary cultures of bovine fetal fibroblast and HEK 293 and Hep G2 cell lines.     

Efficient cold transfection of pea ferredoxin-NADP(H) oxidoreductase into rat hepatocytes

We describe the use of a non-viral, polyethylenimine-based vector to transfect rat hepatocytes preserved under hypothermic storage. DNA sequences encoding Escherichia coli beta-galactosidase and pea ferredoxin-NADP(H) oxidoreductase (FNR), cloned into plasmids pCH110 and pKM4 respectively, were used. FNR was detected in the liver of animals transplanted with transfected cells; no reactivity was observed in endogenous parenchyma. The expression of the transgene was transient as it was detectable up to 96 h subsequently declining to undetectable levels. In contrast to non-transfected cells, the engraftment of FNR-positive cells was not associated with inflammatory reaction. The percentage of FNR-positive implanted hepatocytes was at least five times higher than the original transfection efficiency measured in vitro, while the percentage of beta-galactosidase-positive cells was similar for both methods. These data indicate that the transfection system is effective in the transfer of plasmid DNA into hepatocytes under cold preservation and suggest the advantage of pKM4-transfected hepatocytes on engraftment in the recipient parenchyma.     

Intratumoral spread and increased efficacy of a p53-VP22 fusion protein expressed by a recombinant adenovirus

In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.     

Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops

BACKGROUND: The first objective of the study was to evaluate the transfection of corneal epithelium with non-viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies. METHODS: The human corneal epithelial (HCE) cell line was cultured to different stages of differentiation and transfected with complexes of pCMV-SEAP2 with DOTAP/DOPE, DOTAP/DOPE/protamine sulfate (PS) and polyethylenimine (PEI). The complexes of DOTAP/DOPE with plasmid (CMV-SEAP2 or pCMV-Luc4) were subsequently applied topically to the rabbit eyes. Secreted alkaline phosphatase (SEAP) was analyzed using chemiluminescent assay. Luciferase (Luc) was detected at the mRNA level in cornea and conjunctiva using a qRT-PCR. RESULTS: The transfection levels decreased with differentiation of HCE cells. PEI was effective in transfecting both the dividing and partly differentiated cells, but ineffective in differentiated cells. DOTAP/DOPE showed high activity in differentiated cell cultures, while added PS did not improve transfection. Significant SEAP expression was observed for three days after in vivo transfection in the tear fluid and aqueous humor. The luciferase mRNA was found both in the cornea and conjunctiva. The rates of SEAP secretion from both the basolateral side of differentiated HCE cells and cornea in vivo were within the same range. CONCLUSIONS: Corneal epithelium can be transfected topically to secrete gene products to the tear fluid and aqueous humor. The differentiated HCE model is a useful tool in the evaluation of non-viral carriers for corneal transfection.     

Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen

Recently, self-replicating and self-limiting RNA vaccines (RNA replicons) have emerged as an important form of nucleic acid vaccines. Self-replicating RNA eventually causes lysis of transfected cells and does not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for development of vaccines targeting proteins that are potentially oncogenic. However, the potency of RNA replicons is significantly limited by their lack of intrinsic ability to spread in vivo. The herpes simplex virus type 1 protein VP22 has demonstrated the remarkable property of intercellular transport and provides the opportunity to enhance RNA replicon vaccine potency. We therefore created a novel fusion of VP22 with a model tumor antigen, human papillomavirus type 16 E7, in a Sindbis virus RNA replicon vector. The linkage of VP22 with E7 resulted in a significant enhancement of E7-specific CD8+ T-cell activities in vaccinated mice and converted a less effective RNA replicon vaccine into one with significant potency against E7-expressing tumors. These results indicate that fusion of VP22 to an antigen gene may greatly enhance the potency of RNA replicon vaccines.     

Improving DNA vaccine potency by linking Marek's disease virus type 1 VP22 to an antigen

We have previously employed an intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein to enhance DNA vaccine potency because DNA vaccines lack the intrinsic ability to amplify in cells. Recently, studies have demonstrated that the protein encoded by UL49 of Marek's disease virus type 1 (MDV-1) exhibits some degree of homology to the HSV-1 VP22 protein and features the property of intercellular transport. We therefore generated a DNA vaccine encoding MDV-1 VP22 linked to a model antigen, human papillomavirus type 16 E7. We demonstrated that compared with mice vaccinated with DNA encoding wild-type E7, mice vaccinated with MDV-1 VP22/E7 DNA exhibited a significant increase in number of gamma-interferon-secreting, E7-specific CD8(+)-T-cell precursors as well as stronger tumor prevention and treatment effects. Furthermore, our data indicated that the antitumor effect was CD8 dependent. These results suggested that the development of vaccines encoding VP22 fused to a target antigen might be a promising strategy for improving DNA vaccine potency.     

VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of a VP22-HSVTk-GFP triple fusion construct

BACKGROUND: VP22 is a herpes simplex virus type 1 (HSV-1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22-Tk-GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo. METHODS: VP22-Tk-GFP was cloned into lenti- and adenoviral vectors and used for expression studies, analyses for VP22-mediated protein spreading, and to study the effect of VP22 to thymidine kinase/ganciclovir-mediated cytotoxicity. The function of VP22-Tk-GFP was also investigated in vivo. RESULTS: The triple fusion protein was expressed correctly in vitro, but intercellular trafficking was not observed in any of the studied cell lines. However, under certain conditions, VP22-Tk-GFP sensitized cells more efficiently to ganciclovir than Tk-GFP. In vivo there was a trend for increased inhibition of tumor growth with VP22-Tk-GFP when ganciclovir was present, but the difference with Tk-GFP was not statistically significant. CONCLUSIONS: Based on our results, VP22 fusion proteins do not seem to traffic intercellularly at detectable levels in most tumor cell types. Even though VP22 enhanced cytotoxicity in one cell line in vitro, the effect in vivo was modest. Therefore, our results do not support the utility of VP22 as an enhancer of enzyme prodrug cancer gene therapy.     

Bovine herpesvirus 1 VP22 enhances the efficacy of a DNA vaccine in cattle

For this study, the intercellular trafficking ability of bovine herpesvirus 1 (BHV-1) VP22 was applied to improve the efficacy of a DNA vaccine in calves. A plasmid encoding a truncated version of glycoprotein D (tgD) fused to VP22 was constructed. The plasmid encoding tgD-VP22 elicited significantly enhanced and more balanced immune responses than those induced by a plasmid encoding tgD. Furthermore, protection against a BHV-1 challenge was obtained in calves immunized with the plasmid encoding tgD-VP22, as shown by significant reductions in viral excretion. However, less significant protection was observed for animals vaccinated with the tgD-expressing plasmid, correlating with the lower level of immunity observed prechallenge. This is the first report of the use of VP22 as a transport molecule in the context of a DNA vaccine for a large animal species.     

Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines

Background

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines.

Methods

As the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors.

Results

Combined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression.

Conclusion

Given that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.     

Herpes simplex virus thymidine kinase enzymatic assay in transient transfection experiments using thymidine kinase-deficient cells.

An enzymatic assay for herpes virus simplex type 1 thymidine kinase (HSV-TK) that was sensitive enough to quantitate intracellular levels of enzyme transiently expressed after transfection of HSV-TK vectors into TK-deficient cells using the DNA-calcium phosphate coprecipitation technique is described. TK activity in extracts of transfected cells was determined by binding of [methyl-3H]thymidylate product to thin layers of polyethyleneimine (PEI)-impregnated cellulose. The assay used high-specific-activity [methyl-3H]thymidine as substrate, which required removal of anionic material on a column of PEI-cellulose to enhance the signal-to-noise ratio. The assay was linear over a wide range with respect to the amount of HSV-TK plasmid transfected or content of HSV-TK enzyme in cell extracts. To validate the assay in transient expression experiments, HSV-TK and chloramphenicol acetyltransferase (CAT) plasmids were cotransfected into NIH/3T3 tk- fibroblasts. Transient TK and CAT levels were concordant in cell extracts prepared from replicate plates of transfected cells. Normalizing the transient TK activity for CAT activity from the cotransfected "internal standard" CAT plasmid improved precision significantly, reducing the sample-to-sample coefficient of variation from 41 to 19%. CAT normalization reduced experimental variability mostly by correcting outlying results in transfection efficiency. The HSV-TK reporter gene system based on TK enzymatic assay was thus subject to experimental variation similar to that of the well-established CAT reporter function, demonstrating its utility in transient gene expression analysis.     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

Improving vaccine potency through intercellular spreading and enhanced MHC class I presentation of antigen

The potency of naked DNA vaccines is limited by their inability to amplify and spread in vivo. VP22, a HSV-1 protein, has demonstrated the remarkable property of intercellular transport and may thus provide a unique approach for enhancing vaccine potency. Therefore, we created a novel fusion of VP22 with a model Ag, human papillomavirus type 16 E7, in a DNA vaccine that generated enhanced spreading and MHC class I presentation of AG: These properties led to a dramatic increase in the number of E7-specific CD8(+) T cell precursors in vaccinated mice (around 50-fold) and converted a less effective DNA vaccine into one with significant potency against E7-expressing tumors. In comparison, nonspreading VP22(1-267) mutants failed to enhance vaccine potency. Our data indicated that the potency of DNA vaccines may be dramatically improved through intercellular spreading and enhanced MHC class I presentation of Ag.     

The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression

BACKGROUND: Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. METHODOLOGY: The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. RESULTS AND CONCLUSIONS: In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.