Transcriptional targeting of B cells with viral vectors

In order to optimize viral gene transfer into hematopoietic stem cells we developed retroviral and lentiviral vectors with B cell-specificity. Using fragments of the human CD19 promoter we demonstrate in mice that upon lethal irradiation and reconstitution with virus-treated bone marrow transgene expression is specific for the B cell-lineage. We compare various viral constructs with different promoter length and with or without B cell-specific enhancer regions in retro- and lentiviral backbones. Our data suggest that B cell-targeting for gene therapy approaches is feasible, leads to stable expression, and can be modulated by using different transduction and expression systems.     

Precipitation of nucleotides by calcium phosphate

Earlier it has been shown that nucleic acids of high molecular weight can be introduced into cells by coprecipitation with calcium phosphate. We have studied the requirements for calcium phosphate coprecipitation of shorter nucleotides. The degree of coprecipitation of dodecanucleotides lacking terminal phosphate varied between 25 and 72%. Tetramers with a 5′-monophosphate were coprecipitated to 29–87% by calcium phosphate. A high content of guanosine residues and an increased number of terminal phosphate groups increased the degree of coprecipitation of nucleotides. The trinucleotide pppA2′p5′A2′p5′A was effectively precipitated by calcium phosphate but the monophosphate and the core structure were not.     

Systemic tumor targeting and killing by Sindbis viral vectors

Successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. Although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. By contrast, we show here that blood-borne Sindbis viral vectors systemically and specifically infect tumor cells. A single intraperitoneal treatment allows the vectors to target most tumor cells, as demonstrated by immunohistochemistry, without infecting normal cells. Further, Sindbis infection is sufficient to induce complete tumor regression. We demonstrate systemic vector targeting of tumors growing subcutaneously, intrapancreatically, intraperitoneally and in the lungs. The vectors can also target syngeneic and spontaneous tumors in immune-competent mice. We document the anti-tumor specificity of a vector that systemically targets and eradicates tumor cells throughout the body without adverse effects.     

Permanent genome modifications in plant cells by transient viral vectors

Endonuclease-mediated induction of genomic double-strand breaks has enabled genome editing in living cells. However, deploying this technology for the induction of gene disruption in plant cells often relies on direct gene transfer of endonuclease (i.e. zinc finger nuclease or homing endonuclease) expression constructs into the targeted cell, followed by regeneration of a mutated plant. Such mutants, even when they have no detectable traces of foreign DNA, might still be classified as transgenic because of the transgenic nature of the endonuclease delivery method. Indirect delivery of endonucleases into target cells by viral vectors provides a unique non-transgenic approach to the production of mutated plants. Furthermore, viral vectors can spread into the growing and developing tissues of infected plants, which could provide a unique opportunity to bypass the regeneration step that is often required in direct gene-transfer methods.     

Heterologous encapsidation in transmission of plant viral particles by aphid vectors

Recently, significant progress has been made in recognizing virus-aphid specificities and identifying the proteins involved in virus transmission by aphids. An essential role of the viral capsid protein in this process has been proved. Heterologous encapsidation between viruses in mixed infections may allow transmission by aphids of normally non-aphid-transmissible viruses or change virus-vector interactions. This review describes the most characteristic examples of the phenomenon. Recent findings regarding transmission by aphids of viroid encapsidated in the viral capsid protein, and of virus encapsidated in transgenic coat protein, are presented.     

Immunogenicity of cytopathic and noncytopathic viral vectors

The impact of cytolytic versus noncytolytic viral infections on host responses is not well understood, due to limitations of the systems that have been used to address this issue. Using paired cytopathic and noncytopathic rabies viruses that differ by only two amino acids, we investigated several fundamental aspects of the immune response to these viral vectors. Greater cytopathic capacity translated into a greater degree of cross-priming to CD8(+) T cells (T(CD8)(+)) and more-robust short-term humoral and cellular responses. However, long-term responses to the two viruses were similar, suggesting that direct priming drives the bulk of the T(CD8)(+) antirabies response and that enhanced acute responses associated with greater virally mediated cellular destruction were balanced by other factors, such as prolonged antigen expression associated with noncytopathic virus. Such compensatory mechanisms may be in place to ensure comparable immunologic memories to various pathogens.     

Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate

Artificial casein micelles were prepared by adding 30 mM calcium, 22 mM phosphate and 10 mM citrate to sodium caseinate solutions, and the content of the casein aggregates cross-linked by colloidal calcium phosphate was determined by high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. The content of the casein aggregates cross-linked by colloidal calcium phosphate in artificial whole casein micelles was 48% of total casein, and their relative casein composition determined by high-performance ion-exchange chromatography was 53.1% for alpha s1-casein, 15.8% for alpha s2-casein, 31.1% for beta-casein and 0% for kappa-casein. The order of cross-linking by colloidal calcium phosphate agreed with that of the ester phosphate content of casein constituents. The content of the casein aggregates cross-linked by colloidal calcium phosphate was higher in alpha s1-kappa-casein micelles than in beta-kappa-casein micelles. kappa- and gamma-caseins and dephosphorylated alpha s1-casein were not cross-linked by colloidal calcium phosphate. Although kappa-casein was not cross-linked, chemically phosphorylated kappa-casein, of which the average phosphate content was 8.5 per molecule, was cross-linked. It is concluded that caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate.     

Development of viral vectors and the application for viral entry mechanisms

Virus is identified as one of the obligate intracellular parasites, which only amplify in cells of specific living things. Viral vectors, which are developed by utilizing these properties, are available in the various fields such as basic research of medical biology or application of gene therapy. Our research group has studied development of viral vectors using properties of baculovirus or vesicular stomatitis virus (VSV). Due to the development of new baculoviral vectors for mammalian cells, it is possible to be more efficient transduction of foreign gene in mammalian cells and animals. Furthermore, pseudotype or recombinant VSV possessing the envelope proteins of hepatitis C virus, Japanese encephalitis virus or baculovirus were constructed, and characteristics of the envelope proteins or entry mechanisms of these viruses were analyzed.     

Transfection of adherent and suspended cells by calcium phosphate

DNA-calcium phosphate coprecipitates have been used for 30 years as an efficient method to introduce genetic material into cells. The method involves simple solutions that can be prepared or purchased by the experimentalist. All the numerous variations of the protocol found in the literature are based on the same principle--a spontaneous precipitation that occurs in supersaturated solutions. When DNA is present during this process, it is readily incorporated into the forming calcium phosphate precipitate. Although a wide range of conditions will lead to precipitates, high transfection efficiencies are only obtained within a narrow range of optimized parameters that assure certain properties of the precipitate. This paper describes several physico-chemical parameters that are critical to adapt the method to a particular cell line and/or cultivation condition. Examples of protocols that were established and tested within the authors' laboratory are presented. The article also emphasizes differences between transfections of adherent and suspended cells.     

Biomimetic calcium phosphate mineralization with multifunctional elastin-like recombinamers

Biomimetic hybrid materials based on a polymeric and an inorganic component such as calcium phosphate are potentially useful for bone repair. The current study reports on a new approach toward biomimetic hybrid materials using a set of recombinamers (recombinant protein materials obtained from a synthetic gene) as crystallization additive for calcium phosphate. The recombinamers contain elements from elastin, an elastic structural protein, and statherin, a salivary protein. Via genetic engineering, the basic elastin sequence was modified with the SN(A)15 domain of statherin, whose interaction with calcium phosphate is well-established. These new materials retain the biocompatibility, "smart" nature, and desired mechanical behavior of the elastin-like recombinamer (ELR) family. Mineralization in simulated body fluid (SBF) in the presence of these recombinamers reveals surprising differences. Two of the polymers inhibit calcium phosphate deposition (although they contain the statherin segment). In contrast, the third polymer, which has a triblock structure, efficiently controls the calcium phosphate formation, yielding spherical hydroxyapatite (HAP) nanoparticles with diameters from 1 to 3 nm after 1 week in SBF at 37 °C. However, at lower temperatures, no precipitation is observed with any of the polymers. The data thus suggest that the molecular design of ELRs containing statherin segments and the selection of an appropriate polymer structure are key parameters to obtain functional materials for the development of intelligent systems for hard tissue engineering and subsequent in vivo applications.     

Engineering targeted viral vectors for gene therapy

To achieve therapeutic success, transfer vehicles for gene therapy must be capable of transducing target cells while avoiding impact on non-target cells. Despite the high transduction efficiency of viral vectors, their tropism frequently does not match the therapeutic need. In the past, this lack of appropriate targeting allowed only partial exploitation of the great potential of gene therapy. Substantial progress in modifying viral vectors using diverse techniques now allows targeting to many cell types in vitro. Although important challenges remain for in vivo applications, the first clinical trials with targeted vectors have already begun to take place.     

基因工程疫苗的病毒载体

病毒基因工程疫苗是以活病毒为载体将一段外源基因导入机体细胞内,并使外源基因维持较高水平的表达。通过使用复制型或复制缺陷型载体能使表达的抗原诱生机体产生相应的体液抗体,并能引起机体产生细胞介导的免疫反应及粘膜免疫反应。本文主要介绍有可能用于基因工程疫苗的DNA及RNA病毒载体构建及其应用。