Direct DNA Delivery Research Articles

752. Delivery of Non-Microparticle Naked DNA Vaccine Using Supersonic Flow by a Low-Pressure Gene Gun

DNA vaccines are a new and powerful approach to generation of immunological responses against infectious disease and cancer. DNA can be delivered either into muscle by simple injection or into epidermal by gene gun. Intramuscular injection requires large amount of DNA (100microgram/per mouse) to elicit the immune response; in contrast, microparticlulate bombardment system can induce immune response using very low amount of DNA (1 microgram/per mouse). One disadvantage of gene gun bombardment is that non-biodegradable gold or tungsten may skew the immune response or cause adverse side effect when accumulated. In this report, we have demonstrated the direct delivery of naked DNA without any microparticle using a modified gene gun. The modified gene gun is based on the aerodynamic theory that a supersonic flow is generated when the pressure difference between the inside and the outside pressure of the nozzle is greater than 1.9atm. This high speed airflow can carry the particle from stationary state to accelerate to an extreme high speed (200m/sec). In this way, the naked DNA may be directly transformed into the mammalian cells. Previous study indicated that this modified gene gun achieve similar deliver efficacy as the gene gun commercially available using gold-coated DNA. In this report, we showed that the modified gene gun can achieve 10–40% delivery efficacy as the gold particle coated with DNA in Balb/C mice, C57BL/6 mice, and C3H mice using luciferase gene reporter driven by CMV promoter. Then, we examined the immune responses elicited by inoculation of DNA encoding HBV large surface protein expressed by its own promoter. The non-microparticle DNA and gold-coated DNA elicited similar humoral immunity as shown by antibody titer. Similar overall cytokine profile was induced by either type of DNA vaccine. Furthermore, we examined the therapeutic responses with the DNA vaccine against the extracellular domain of neu on the mouse tumor (MBT-2) naturally overexpressing neu in C3H mice. The results indicated that non-microparticle naked neu DNA vaccine can achieve similar anti-tumor effect as the gold-coated neu DNA vaccine in C3H mice at the dose of 1 microgram/per mouse. Ying-Chang Wang is an employee of BioWare Technologies Co., Ltd, Taipei, Taiwan.

Multilayered polyelectrolyte films promote the direct and localized delivery of DNA to cells

Multilayered polyelectrolyte films fabricated from plasmid DNA and a hydrolytically degradable synthetic polycation can be used to direct the localized transfection of cells without the aid of a secondary transfection agent. Multilayered assemblies 100 nm thick consisting of alternating layers of synthetic polymer 1 and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) were deposited on quartz substrates using a layer-by-layer fabrication procedure. The placement of film-coated slides in contact with COS-7 cells growing in serum-containing culture medium resulted in gene expression in cells localized under the film-coated portion of the slides. The average percentage of cells expressing EGFP relative to the total number of cells ranged from 4.6% to 37.9%, with an average of 18.6% ± 8.2%, as determined by fluorescence microscopy. In addition to providing a mechanism for the immobilization of DNA at the cell/surface interface, a preliminary analysis of film topography by atomic force microscopy (AFM) demonstrated that polymer 1 /DNA films undergo significant structural rearrangements upon incubation to present surface bound condensed plasmid DNA nanoparticles. These data suggest that the presence of the cationic polymer in these materials may also contribute to the internalization and expression of plasmid. The materials and design principles reported here present an attractive framework for the local or non-invasive delivery of DNA from the surfaces of implantable materials or biomedical devices.

202. DNA Delivery to the Mitochondria Sites Using Leader Peptide Conjugated Polyethylenimine

Background: Some genetic diseases are associated with defects of the mitochondrial genome. Mitochondria gene therapy by direct DNA delivery to mitochondria has been suggested as a therapeutic option for the diseases. However, a few studies about DNA delivery to mitochondria has been reported and no definite delivery method is available. Although numerous viral and non-viral gene carriers have been developed, these carriers were designed for the delivery of DNA into the nucleus. Thus, mitochondrial gene therapy has been thought as theoretical and speculative. In this research, we hypothesized that mitochondrial leader peptide (LP) conjugated polyethylenimine (PEI) could deliver DNA to the mitochondrial sites.

Transgene integration in plants: poking or patching holes in promiscuous genomes?

Transgene integration in plants transformed by either Agrobacterium or direct DNA delivery methods occurs through illegitimate recombination (IR). The precise mechanism(s) for IR-mediated transgene integration and the role of host double-strand break repair enzymes remain unknown. A recent wealth of sequenced transgene loci and investigations aimed at genetically dissecting transgene integration mechanism(s) have provided new insights into the process.

Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment.

A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery. However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods. The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis. In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced. Both loci exhibited rearranged delivered DNA and flanking genomic sequences. The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site. Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus. Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs. The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci. The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity.

Invasive drug delivery.

The central nervous system is a very attractive target for new therapeutic strategies since many genes involved in neurological diseases are known and often only local low level gene expression is required. However, as the blood brain barrier on one hand prevents some therapeutic agents given systematically from exerting their activity in the CNS, it also provides an immune privileged environment. Neurosurgical technology meanwhile allows the access of nearly every single centre of the CNS and provides the surgical tool for direct gene delivery via minimal invasive surgical approaches to the brain. Successful therapy of the central nervous system requires new tools for delivery of therapeutics in vitro and in vivo (Fig. 1). The application of therapeutic proteins via pumps into the CSF was shown to be only of limited value since the protein mostly is not sufficiently transported within the tissue and the half life of proteins limits the therapeutic success. Direct gene delivery into the host cell has been a main strategy for years, and in the beginning the direct DNA delivery or encapsulation in liposomes or other artificial encapsulation have been applied with different success. For several years the most promising tools have been vectors based on viruses. Viruses are able to use the host cell machinery for protein synthesis, and some of them are able to stably insert into the host cell genome and provide long term transgene expression as long as the cell is alive. The increasing knowledge of viruses and their live cycle promoted the development of viral vectors that function like a shuttle to the cell, with a single round of infection either integrating or transiently expressing the transgene. Viral vectors have proven to be one of the most efficient and stable transgene shuttle into the cell and have gained increasing importance. The limitations of some viral vectors like the adenoviral vector and adeno-associated viral vector have been improved by new constructs like HIV-1 based lentiviral vectors. The immune response caused by expression of viral proteins, or the inability of some viral vectors like the retroviral vector to infect only dividing cells have been overcome by these new constructs. Lentiviral vectors allow an efficient and stable transgene expression over years in vivo without effecting transgene expression or immune response. In this Chapter we will describe synthetic vectors, give an overview of the most common viral vectors and focus our attention on lentiviral vectors, since we consider them to be the most efficient tool for gene delivery in the CNS.

Nonsurgical direct delivery of plasmid DNA into rat heart: time course, dose response, and the influence of different promoters on gene expression.

Transfer of genes encoding therapeutic proteins into the myocardium shows great potential for treatment of coronary artery disease. To quantitatively elucidate the behavior of plasmid DNA following cardiac gene transfer, time kinetics, dose-response relationship, systemic spread to the liver, and the influence of different promoters on plasmid DNA gene expression in rat hearts were examined using a novel nonsurgical direct delivery method that enables testing of large numbers of animals. Plasmids encoding either vascular endothelial growth factor A 165 or a fusion protein between enhanced green fluorescent protein (EGFP) luciferase were injected directly in rat hearts under echocardiographic guidance. The results show that gene expression is dose related and that the duration of gene expression is transient. These findings underscore the necessity to explore other efficient vectors or alternative methods of gene delivery to achieve increased and prolonged gene expression.

Anti-idiotypic DNA vaccines for B-cell lymphoma therapy.

The idiotypic determinants expressed by immunoglobulin at the surface of malignant B-cells provide specific targets for vaccination strategies. However, as self-antigens they are poorly immunogenic and vaccines must include carriers to improve immune responses. Chemical cross-linking of purified idiotypic protein is so far the only method which has been employed in clinical trials while a number of second-generation vaccines have been developed in mouse models. These strategies are based on the use of recombinant DNA technology to create fusion proteins that contain the idiotype genetically linked to molecules that act as immunological adjuvants. Fusion proteins can be made available to the immune system by direct delivery of naked DNA with great advantages in terms of time and costs. The most relevant approaches are listed and discussed with particular emphasis to the mechanism by which different molecules exert their adjuvant effect. The role of cellular versus antibody mediated suppression of tumor growth following Id vaccination is examined by comparing the most recent data on the mechanism of tumor protection. In addition, an analysis of the specificity of anti-idiotypic antibodies induced by scFv-DNA vaccination as compared to protein immunization is provided.

Direct DNA delivery into zebrafish embryos employing tissue culture techniques.

The production of transfected fish embryos requires expertise in injecting the fertilized eggs and/or expensive equipment for electroporation or microprojectiles. This article demonstrates that by exposure to DNA constructs conjugated with transfecting reagents dechorionated Danio rerio embryos are capable of acquiring extracellular DNA and expressing reporter genes. Embryos incubated with pCMVluc complexed with GeneJammer or GenePORTER expressed luciferase 24-48 h after exposure. pCMVGFP DNA mixed with the same agents generated embryos that exhibited differential patterns of expression of green fluorescent protein (GFP). Embryonic development varied depending on the procedure employed and the reporter gene utilized. Expression of the luciferase gene did not interfere with the subsequent development of the embryos. In contrast, the embryos expressing a high level of GFP were affected, probably due to a very active promoter. These results demonstrate the ease of obtaining transfected fish embryos, which facilitate the mass production of new genotypes and extend the procedure to laboratories with limited resources.

Use of Wild Arachis Species/Introgression of Genes into A. hypogaea L.

Abstract The use of wild Arachis L. in cultivar improvement programs has been considered an option for more than 50 yr. Both A. Krapovickas and W.C. Gregory, independently, made interspecific hybridizations in the 1940s. However, only three cultivars have been released as a result of interspecific hybridizations, and only one of those has a clearly identifiable genetic component from the wild species. Several breeding lines have been reported and several germplasm releases are documented from Texas, North Carolina, and ICRISAT. At least four potential options exist for transferring genes from wild Arachis to the cultigen: a) The hexaploid pathway consists of crossing a diploid wild species directly with A. hypogaea, doubling the chromosome number to the hexaploid level, then backcrossing for several generations to restore the tetraploid condition. Several options are possible in this pathway involving various crossing schemes prior to crossing a diploid hybrid with A. hypogaea. North Carolina and ICRISAT have had success with this pathway. b) The diploid/tetraploid pathway has been the most successful in Texas to date. This pathway involves crossing diploid species (two to several), doubling the chromosome number of the hybrid, then crossing to A. hypogaea and backcrossing with selection for the desired character. This pathway is most successful when both A-and B-genome species are involved. Germplasm lines and a cultivar have been released in Texas using this pathway. c) Another diploid/tetraploid pathway could be to double chromosome numbers of diploid species and cross the amphiploids directly with A. hypogaea. Several attempts have been made with this technique, but no germplasm releases have been reported, in large part because sterility is too great when both A and B genomes are not included in the hybrid. Many of the sections/species of wild Arachis are so greatly isolated from A. hypogaea that plant transformation will be the likely method to introduce genes into the cultigen. d) Molecular methods of “inserting” genes into peanut that have been modestly successful and include use of Agrobacterium spp., electroporation, and direct DNA delivery techniques such as the gene gun, whiskers, and sonication. No releases have resulted.

Bacterial systems for the delivery of eukaryotic antigen expression vectors.

Attenuated bacterial strains allow the administration of recombinant vaccines via the mucosal surfaces. Whereas attenuated bacteria are generally engineered to express heterologous antigens, a novel approach employs intracellular bacteria for the delivery of eukaryotic antigen expression vectors (so-called DNA vaccines). This strategy allows a direct delivery of DNA to professional antigen-presenting cells (APC), such as macrophages and dendritic cells (DC), through bacterial infection. The bacteria used for DNA vaccine delivery either enter the host cell cytosol after phagocytosis by the APC, for example, Shigella and Listeria, or they remain in the phagosomal compartment, such as Salmonella. Both intracellular localizations of the bacterial carriers seem to be suitable for successful delivery of DNA vaccine vectors.

Whisker-mediated transformation of embryogenic callus of maize.

The present study was designed to establish embryogenic callus as a target tissue for whisker-mediated transformation of maize (Zea mays L.). Silicon carbide whiskers were used to deliver the bar and uidA (GUS) genes into embryogenic maize callus. Samples of osmotically-treated Type II callus were vigorously agitated in the presence of whiskers and plasmid DNA using a standard laboratory vortex or a modified dental amalgamator. On average, three transgenic callus lines were obtained per 100 samples treated. Plants were regenerated from several GUS-expressing callus lines and DNA analyses confirmed stable integration and inheritance. As with other direct DNA delivery methods involving embryogenic maize callus, integration patterns of the inserted DNA appeared to be complex. Although currently less efficient than microparticle bombardment on a per target basis, whisker-mediated transformation of embryogenic callus represents a viable method for transgenic maize production.

Electrically mediated plasmid DNA delivery to hepatocellular carcinomas in vivo.

Gene therapy by direct delivery of plasmid DNA has several advantages over viral gene transfer, but plasmid delivery is less efficient. In vivo electroporation has been used to enhance delivery of chemotherapeutic agents to tumors in both animal and human studies. Recently, this delivery technique has been extended to large molecules such as plasmid DNA. Here, the successful delivery of plasmids encoding reporter genes to rat hepatocellular carcinomas by in vivo electroporation is demonstrated.

Alphavirus vectors: from protein production to gene therapy

Alphaviruses are enveloped viruses containing a single positive strand RNA molecule as genome. Several vectors derived from alphaviruses have been developed, which include Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE). Alphavirus self-replicating RNA containing heterologous genes can be synthesized in vitro from plasmids having the recombinant alphavirus replicon sequences under the control of a prokaryotic promoter, such as SP6 or T7. High level expression of the heterologous proteins/RNA is obtained in cells transfected with this RNA. Although the system can be used for gene delivery directly as naked RNA, several packaging systems have been developed which allow the encapsidation of the alphaviral recombinant RNA into suicidal viral particles, increasing the efficiency of delivery of the recombinant genome into cells. A more recent variant of the system is based on a DNA/RNA layered alphaviral vector in which the recombinant replicon is transcribed from an RNA polymerase-II promoter, allowing direct delivery of DNA into cells. Alphavirus vectors have been used to express a great number of proteins with many different purposes, including protein production and characterization, functional studies, vaccination, and gene therapy. Both the recombinant alphavirus particles and the alphavirus nucleic acid vectors have shown to be able to induce protective immune responses in animal models. The possible application of these vectors in gene therapy faces, however, two limitations, which are the lack of specific targeting and the transient nature of the vector, due to the induction of apoptosis by the alphavirus replicon. Several strategies have been recently described to improve the targeting of alphavirus vectors and to develop noncytopathic vectors with potential use in gene therapy.

Plant transformation technology. Developments and applications.

Plant transformation has its roots in the research on Agrobacterium that was being undertaken in the early 1980s. The last two decades have seen significant developments in plant transformation technology, such that a large number of transgenic crop plants have now been released for commercial production. Advances in the technology have been due to development of a range of Agrobacterium-mediated and direct DNA delivery techniques, along with appropriate tissue culture techniques for regenerating whole plants from plant cells or tissues in a large number of species. In addition, parallel developments in molecular biology have greatly extended the range of investigations to which plant transformation technology can be applied. Research in plant transformation is concentrating now not so much on the introduction of DNA into plant cells, but rather more on the problems associated with stable integration and reliable expression of the DNA once it has been integrated.

Chromosome-mediated and direct gene transfers in wheat

Wild grasses, including relatives of wheat, have several desirable characters that can be introduced into both bread wheat and durum wheat. Since current wheat cultivars lack certain traits, for example, resistance to fusarium head blight (scab), related wild grasses may be the only option for useful variability. Wide hybridization of wheat with grasses, coupled with cytogenetic manipulation of the hybrid material, has been instrumental in the genetic improvement of wheat. Chromosome engineering methodologies, based on the manipulation of pairing control mechanisms and induced translocations, have been employed to transfer into wheat specific disease and pest resistance genes from annual (e.g., rye) or perennial (e.g., Thinopyrum spp., Lophopyrum spp., and Agropyron spp.) members of the wheat tribe, Triticeae. The advent of in situ hybridization techniques, for example, fluorescent GISH combined with Giemsa C-banding, has proved immensely useful in characterizing alien chromatin specifying resistance to various pathogens and pests. The use of DNA markers (RAPDs and RFLPs) helps to identify desirable genotypes more precisely and, thereby, facilitates gene transfer into wheat. Such markers may be particularly helpful in monitoring the introgression of alien genes in the wheat genome. In fact, several cultivars, particularly of bread wheat, contain superior traits of alien origin. The development of novel gene-transfer techniques in the past decade that allow direct delivery of DNA into regenerable embryogenic callus of wheat has opened up new avenues of alien-gene transfer into wheat cultivars. Thus, transgenic bread and durum wheats have been produced and methods of gene delivery standardized. The application of transgenic technology has not only yielded herbicide-resistant wheats, but has also helped to improve grain quality by modifying the protein and starch profiles of the grain. These in vitro approaches to gene transfer are developing rapidly, and promise to become an integral part of plant breeding efforts. However, the new biotechnological tools will complement, not replace, conventional plant breeding.Key words: alien-gene transfer, fluorescent GISH, Giemsa banding, homoeologous chromosome pairing, molecular markers, transgenic bread wheat, transgenic durum wheat.

Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Switchgrass

Embryogenic cell suspension cultures represent an enhanced culture type for the selection of mutants, mass propagation, direct delivery of DNA in to intact regenerable cells and isolation of totipotent protoplasts. The objectives of this study with switchgrass (Panicum virgatum L.) were to establish embryogenic suspension cultures and to regenerate plants from these cultures. The cultures were initiated from embryogenic callus obtained from young inflorescences. They were maintained in Murashige and Skoog liquid medium containing 9.0 μM 2,4‐dichlorophenoxyacetic acid (2,4‐D), 4.4 μM 6‐benzylaminopurine (BAP) and 30 g ‐1 maltose. The s uspension was passed througha 210‐μm mesh and was comprised mainly of small, round densely cytoplasmic starch containing cells with distinct nuclei. Somatic embryos developed from these cells directly and were free floating in the medium. A sequence of developmental stages from embryogenic cells to fully developed embryos was observed in the cultures. The embryos had the typical morphology of a grass embryo. Embryogenic calluses were also obtained 4 wk after the suspension was plated onto solid medium containing various combinations of 2,4‐D and BAP. Embryos from suspension and suspension‐derived callus cultures germinated to produce plants that were successfully established in the greenhouse.

Gene transfer vectors based on Sendai virus

A gene delivery system is a fundamental technology used in human gene therapy. In order to treat patients suffering from incurable metabolic diseases, we must be able to deliver genes efficiently in situ and induce stable gene expression in non-dividing tissue cells. However, none of the current gene transfer systems (both viral and non-viral) satisfies this goal. In order to develop a novel gene delivery system that is free from the defects of existing gene transfer vectors, we analyzed natural biological phenomena that involve gene transfer and expression, and made artificial components that mimic the functioning of these systems. Our recent results shed light on three major aspects of gene transfer and expression: (1) the direct delivery of DNA into cytoplasm using fusogenic liposomes, (2) the transfer of DNA from cytoplasm to nucleus with a nuclear localization signal, and (3) the stabilization of DNA in the nucleus as an independent replicon. The possible development of a hybrid vector by combining these components is discussed.

Transformation of peanut with a soybean vspB promoter-uidA chimeric gene. I. Optimization of a transformation system and analysis of GUS expression in primary transgenic tissues and plants.

Direct DNA delivery via microprojectile bombardment has become an established approach for gene transfer into peanut (Arachis hypogaea L.). To optimize our transformation protocol and to simultaneously explore the function of a heterologous promoter whose activity is developmentally regulated, embryogenic cultures from three peanut cultivars were bombarded with two plasmid constructs containing a uidA gene controlled by either a soybean vegetative storage protein gene promoter or a cauliflower mosaic virus 35S promoter. We found that GUS transient expression was useful to predict stable transformation and confirmed that image analysis could provide a quick and efficient method for semi-quantitation of transient expression. One hundred and sixty hygromycin-resistant cell lines were recovered from and maintained on selective medium, and those tested by Southern blot analysis showed integration of the foreign gene. Over 200 transgenic plants were regenerated from 38 cell lines. More than 100 plants from 32 cell lines flowered and 79 plants from 19 cell lines produced pods. Over 1000 R1 seeds were harvested. Analysis of expression in primary transgenic plants showed that GUS expression driven by the vspB promoter was modulated by chemical and positional information.

Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.

Successful gene therapy approaches will require efficient gene delivery and sustained expression of the transgene in recipients. A variety of methods, ranging from direct DNA delivery to infection with recombinant viruses containing foreign genes, have been developed, but they all have some major limitations that restrict their utility. We have described a human lentiviral (HIV)-based vector that can transduce non-dividing cells in vitro and deliver genes in vivo. With this vector, expression of transgenes in the brain has been detected for more than six months--the longest period tested so far. Because lentiviral vectors are pseudotyped with vesicular stomatitis virus G glycoprotein (VSVG; ref. 8), they can transduce a broad range of tissues and cell types. We now describe the ability of lentiviral vectors to introduce genes directly into liver and muscle. Sustained expression of green fluorescent protein (GFP), used as a surrogate for therapeutic protein, can be observed for more than 22 weeks in the liver. Similar long-term expression (more than eight weeks) was observed in transduced muscle. In contrast, little or no GFP could be detected in liver or muscle transduced with the Moloney murine leukaemia virus (M-MLV), a prototypic retroviral based vector. At a minimum, 3-4% of the total liver tissue was transduced by a single injection of 1-3 x 10(7) infectious units (I.U.) of recombinant HIV vector. Furthermore, no inflammation of recruitment of lymphocytes could be detected at the site of injection. Animals previously transduced with a lentiviral vector can be efficiently re-infected with lentiviral vectors. Additionally, we show that the requirement for lentiviral accessory proteins to establish efficient transduction in vivo is tissue dependent.