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PubMedDate not supplied

Receptor-mediated gene delivery into antigen presenting cells using mannosylated chitosan/DNA nanoparticles.

Dendritic cells (DCs) are professional antigen presenting cells that induce, sustain, and regulate immune responses. Gene modification of DCs is of particular interest for immunotherapy of diseases where the immunes system has failed or is abnormally regulated, such as in cancer or autoimmune disease. Gene transfer using non-viral vectors is a promising approach for the safe delivery of therapeutic DNA. Among various non-viral vectors, chitosan is considered to be a good candidate for gene delivery system, however, lack of cell specificity and low transfection of chitosan need to be overcome prior to clinical use. In this study, mannosylated chitosan (MC) was prepared to induce the receptor-mediated endocytosis and targeting into antigen presenting cells (APCs), especially DCs having mannose receptors. MC showed great ability to form complexes with DNA and showed suitable physicochemical properties for gene delivery system. It had low cytotoxicity and exhibited much enhanced gene transfer efficiency on the macrophage cell line than chitosan itself. Also, MC/DNA complex was more efficient for transferring IL-12 gene into DCs rather than water-soluble chitosan (WSC)/DNA one, which resulted in better induction of INF-gamma from DCs. Therefore, MC is a promising gene delivery system for repeated administration to maintain sustained gene expression, thereby opening the possibility for immunotherapy.

Animals↗
PubMed2000

Development of oral vaccines for human use.

In developed and developing countries, oral vaccine formulations that elicit protection at mucosal surfaces are attractive vaccine candidates. Research has shown that vaccine delivery using either viral or non-viral vector delivery of heterologous proteins via the oral route is highly effective. Improvements in non-viral vector uptake, specific targeting, antigen presentation and antigen release times will be required to overcome differences in the immune response following delivery. In contrast, the use of plant and animal viruses as vectors provides an effective method for targeted gene delivery. Recently, recombinant plant viruses grown in plants or transgenic plants have been proposed as edible vaccines for human use. Food plants offer many advantages as affordable, oral vaccines, especially for use in developing countries. Further research is necessary to develop strategies to improve immunological memory following oral vaccination and to avoid immunological tolerance in the host.

Adenoviridae↗
PubMed2002

Soluble coxsackievirus adenovirus receptor is a putative inhibitor of adenoviral gene transfer in the tumor milieu.

PURPOSE: Several barriers that collectively restrict gene delivery by viral vectors in vivo have been described. Previously, we identified soluble chondroitin sulfate-proteoglycans/glycosaminoglycans in malignant pleural effusions (MPEs) as inhibitors of retroviral vector transduction. Soluble components of MPE also inhibited adenoviral (Ad) gene transfer, and the factors were characteristically filterable, titrable, stable at 56 degrees C, and blocked the binding of Ad to target cells. Depleting immunoglobulin from MPE, partially reversed the block to Ad transduction, instigating a search for additional factors that bound Ad in MPE. EXPERIMENTAL DESIGN: Vector-protein interactions were identified after the resolution of MPE-components by SDS-PAGE. Viral overlays and immunoblots delineated significant interactions, and the potential relevance of those interactions was tested in transduction efficiency bioassays. RESULTS: Immunoglobulin is the predominant factor inhibiting Ad gene transfer in MPE. Albumin also interacted with Ad, although at predicted serum concentrations, it did not effect Ad transduction efficiency in vitro. Soluble coxsackievirus-Ad receptor (sCAR) was then identified in MPE. In a survey of 18 MPE, the mean concentration of sCAR was variable and estimated to be 3.51 +/- 5.02 ng/ml by ELISA. The impact of sCAR on transduction efficiency in this milieu was next assessed. Whereas immunodepletion of sCAR from MPE by affinity chromatography resulted in enhanced gene transfer within MPE, the inhibition of adenoviral gene transfer was not evident when the predicted concentrations of recombinant sCAR were added into the transduction medium. CONCLUSIONS: These studies indicate that, in addition to anti-Ad antibodies, other specific and nonspecific factors interact with viral vectors and may impair gene transfer in the tumor milieu. The presence of sCAR in MPE puts forward the notion that in certain contexts (e.g., within the extracellular matrix of solid tumors) the concentrations of secreted (or shed) CAR may be high enough to effectively compete with Ad gene delivery.

Adenoviridae↗
PubMed2002

Experimental and therapeutic approaches to muscular dystrophies.

PURPOSE OF REVIEW: Most patients suffering from muscular dystrophies can now obtain a precise diagnosis of their underlying molecular defect, but no efficient treatment to prevent disability and death. This review summarizes recent progress towards developing efficient treatments for these severe diseases. RECENT FINDINGS: Different levels of progress have been achieved in three main approaches: gene therapy, cell therapy and pharmacological therapy. Gene therapy has progressed by improving different vectors for gene delivery. Adenoviruses (mainly high capacity versions) and adeno-associated viruses were the most explored viral vectors. Progress was made in understanding the factors needed for an efficient transfection of muscle. An understanding of protein structure and function in muscular dystrophies has allowed elegant examples of protein engineering as a way of gene therapy. Non-viral vectors for gene transfer, targeted gene modification and transcription modulation have also been explored recently. Cell therapy (myogenic-cell transplantation) progressed in understanding myoblast transplantation in primates for human applications, evaluating protocols for the control of graft rejection, understanding the biology of donor myogenic cells, and searching for alternative sources of donor cells. Three clinical trials using pharmacological approaches (anabolic agents and gentamicin) show very poor or negative results. Other pharmacological approaches (upregulation of alternative therapeutic proteins) are still being researched in mice. SUMMARY: This panoply of experimental approaches covered all the current possibilities of attacking the problem of treating muscular dystrophies. It is expected that one or more will progress to provide efficient tools for the ultimate clinical goal: to prolong function and life in severe muscular dystrophy patients.

Animals↗
PubMed2001

Gene therapy strategies for the treatment of chronic viral hepatitis.

Chronic viral hepatitis is a major clinical problem, with over half a billion persons infected worldwide. Current therapies, principally treatment with recombinant IFN-alpha protein, have limited benefit. Recent studies suggest that gene-based expression of IFN-alpha is a possible therapeutic alternative that may improve the effectiveness of treatment. Gene delivery to the liver and consequent IFN-alpha expression therein, has the potential to concentrate the protein at the target organ and provide more continuous exposure to the therapeutic agent. Other potential gene and nucleic acid therapeutics for viral hepatitis are also being investigated. Key to the deployment of these future therapies is a suitable method of gene delivery. Although recombinant viral vector systems, such as adenovirus, are currently the most effective means of gene delivery to the liver, their use presents many concerns. These include immune and inflammatory reactions to the viral vector and possible adverse interactions between the recombinant virus and the pre-existing viral infection. Non-viral gene delivery systems would be a preferred treatment modality. The efficiency of current non-viral systems is not adequate for systemically administered liver gene therapy. However, recent use of membrane permeabilisation techniques has shown that high efficiency non-viral gene transfer agents are possible. The future coupling of these improved delivery systems with gene- or nucleic acid-based therapeutics currently in development holds out great promise for new generations of antihepatitis therapies.

Animals↗
PubMed2002

In vivo electrotransfer of the cardiotrophin-1 gene into skeletal muscle slows down progression of motor neuron degeneration in pmn mice.

Among all vectors designed for gene therapy purposes, adenovirus appears to be the most efficient in vivo vehicle to transduce the broadest spectrum of cellular targets. However, the deleterious immunogenicity of this viral vector impedes its use in chronic diseases. Non-viral vectors, such as naked DNA, are attractive alternatives for safety and technical issues, such as scale-up production. Naked DNA injection, greatly improved when combined with electroporation, showed great potential in adult animals, especially when directed to the muscle. We have recently proven the therapeutic effect of a neonatal single intramuscular injection of a cardiotrophin-1 (CT-1)-encoding adenovirus in a hereditary disease mouse model of human motor neuron disease, the progressive motor neuronopathy (pmn) mutant. We now demonstrate that a single injection/electroporation of a CT-1-encoding plasmid in neonate pmn mice is almost as efficient as adenovirus-mediated gene transfer with respect to survival, muscular function and neuroprotection of the animals. Treated mice gain global weight, their mean lifespan is extended by 25%, all their electromyographic parameters are improved and myelinated axons of their phrenic nerves are protected. Moreover, we show that re-injection/electroporation leads to improvements in this neuroprotection. We therefore demonstrate for the first time the therapeutic efficacy of neonatal intramuscular DNA injection/electroporation in a murine model of a human hereditary disorder.

Animals↗
PubMed2000

Herpesvirus saimiri-based gene delivery vectors maintain heterologous expression throughout mouse embryonic stem cell differentiation in vitro.

In order to achieve a high efficiency of gene delivery into rare cell types like stem cells the use of viral vectors is presently without alternative. An ideal stem cell gene therapy vector would be able to infect primitive progenitor cells and sustain or activate gene expression in differentiated progeny. However, many viral vectors are inactivated when introduced in developing systems where cell differentiation occurs. To this end, we have developed a mouse in vitro model for testing herpesvirus saimiri (HVS)-based gene therapy vectors. We demonstrate here for the first time that HVS is able to infect totipotent mouse embryonic stem (ES) cells with high efficiency. We have transduced ES cells with a recombinant virus carrying the enhanced green fluorescent protein (EGFP) gene and the neomycin resistance gene (NeoR) driven by a CMV promoter and the SV40 promoter, respectively. ES cells maintain the viral episomal genome and can be terminally differentiated into mature haematopoietic cells. Moreover, heterologous gene expression is maintained throughout in vitro differentiation. Besides its obvious use in gene therapy, this unique expression system has wide ranging applications in studies aimed at understanding gene function and expression in cell differentiation and development.

Animals↗
PubMed2002

Polyethylenimine Derivatives as Potent Nonviral Vectors for Gene Transfer.

The delivery of a functional gene into a tissue can allow the correction of gene defaults or mutations such as those observed in severe hereditary pathologies or in cancer tissues and could lead to gene therapy. To achieve gene transfer, viral vectors are mainly used because of their intrinsic ability to enter the cells and promote expression of the transgene. However, many factors can limit the use of viral vectors, including a heavy laboratory infrastructure, and, in the case of iterative administration, the induction of immune response against viral proteins. Alternative gene transfer technologies based on nonviral vectors have been proposed. Polyethylenimine (PEI) derivatives are polycationic molecules that are able to form stable complexes with plasmidic DNA. PEI/DNA complexes attach to the cell surface, migrate into clumps that enter the cell by endocytosis and are deagregated in an acidic lysosomal compartment and/or enter the nucleus. PEI derivatives can be proposed as linear (22 kDa) or reticulated (25 kDa) molecules that prove efficient for gene transfer in vitro and in vivo. Besides extensive applications of unsubstituted PEI, glycosylated-PEI derivatives were proposed and reported to enhance gene transfer efficiency through decreased size and aggregation of PEI/DNA complexes. Galactosylated-PEI derivatives have been reported to enhance interactions with cell membranes through carbohydrate-binding protein recognition and specifically target PEI/DNA complexes toward biological systems that express galectins. More recently, glucosylated PEI derivatives have been shown to yield higher and longer-lasting transgene expression than unsubstituted-PEI in human head and neck carcinoma tumor cells. In the present paper, a review of in vitro and in vivo properties of PEI-mediated gene transfer experiments is presented. (c) 2002 Prous Science. All rights reserved.

Journal Article↗
PubMed2003

Tetracycline-inducible transgene expression mediated by a single AAV vector.

Regulated gene delivery systems are usually made of two elements: an inducible promoter and a transactivator. In order to optimize gene delivery and regulation, a single viral vector ensuring adequate stoichiometry of the two elements is required. However, efficient regulation is hampered by interferences between the inducible promoter and (i) the promoter used to express the transactivator and/or (ii) promoter/enhancer elements present in the viral vector backbone. We describe a single AAV vector in which transcription of both the reverse tetracycline transactivator (rtTA) and the transgene is initiated from a bidirectional tetracycline-responsive promoter and terminated at bidirectional SV40 polyadenylation sites flanking both ITRs. Up to 50-fold induction of gene expression in human tumor cell lines and 100-fold in primary cultures of rat Schwann cells was demonstrated. In addition an 80-fold induction in vivo in the rat brain has been obtained. In vitro, the autoregulatory vector exhibits an induced expression level superior to that obtained using the constitutive CMV promoter. Although extinction of the transgene after removal of tetracycline was rapid (less than 3 days), inducibility after addition of tetracycline was slow (about 14 days). This kinetics is suitable for therapeutic gene expression in slowly progressive diseases while allowing rapid switch-off in case of undesirable effects. As compared to previously described autoregulatory tet-repressible (tetOFF) AAV vectors, the tet-inducible (tetON) vector prevents chronic antibiotic administration in the uninduced state.

Animals↗
PubMed2002

Genetically modified adenovirus vector containing an RGD peptide in the HI loop of the fiber knob improves gene transfer to nonhuman primate isolated pancreatic islets.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post-transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the alphavbeta3 and alphavbeta5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate Pis were isolated by a semi-automated technique. Steptozotocin-induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (>80% of the islet cells were infected at 10 particle-forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP).More than 90% of the infected cells were insulin-producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose-stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80% transfection efficiency) compared with AdLuc at high titers. Finally, long-term euglycemia (>250d) was observed in 89% of the animals that received PI infected with AdRGDLuc compared with none of the animals that received PI infected with AdLuc. The present study provides new information about the possibility of tropism modification of Ad vectors to increase the transfection efficiency and transgene expression to isolated PI. Incorporation of the RGD sequence in the HI loop of the fiber knob allows highly efficient transfection efficiency to nonhuman primate insulin-producing cells and adequate long-term function of the p-cell after transplantation.

Adenoviridae↗
PubMed2005

Intratumoral injection of dendritic cells transduced by an SV40-based vector expressing interleukin-15 induces curative immunity mediated by CD8+ T lymphocytes and NK cells.

Cancer immunotherapy has been extensively attempted by gene transfer of cytokines with viral vectors. In this work, we compared the therapeutic effects of interleukin 12 and 15 (IL-12 and IL-15) genes transferred to tumor cells or to dendritic cells (DCs), which were subsequently injected into established tumors. For this purpose, we used viral vectors based on simian virus 40 (rSV40). Importantly, we observed that nonmatured DCs infected with rSV40 vectors remained phenotypically immature. Infection of CT-26 tumor cells with rSV40 expressing IL-12 (rSVIL-12) or IL-15 (rSVIL-15) failed to inhibit tumor development. In contrast, the intratumoral administration of syngeneic DCs transduced with rSVIL-12 or rSVIL-15 was associated with a strong antitumor response; up to 40% tumor remissions were achieved with DCs transduced by rSVIL-12 and 73% with DCs expressing IL-15. This antitumor effect correlated with the in vivo priming of tumor-specific CD8+ T lymphocytes. Depletion studies showed that rSVIL-15-mediated antitumor efficacy was mediated mainly by CD8+ T lymphocytes and NK cells. We conclude that (i) SV40-derived vectors are an advantageous alternative to transduce genes into DCs and (ii) DCs transferred with IL-15 have an enhanced capability to induce curative antitumor immunity when injected into malignant lesions.

Adenocarcinoma↗
PubMed2006

Increased expression of 5-HT1B receptors in rat nucleus accumbens via virally mediated gene transfer increases voluntary alcohol consumption.

Serotonin 5-HT(1B) receptors have been linked to alcoholism in humans and alcohol consumption in rodents. We hypothesize that these receptors, which are located on the axon terminals of nucleus accumbens' (NAcc) projection neurons, modulate alcohol reward mechanisms. To test this hypothesis, we measured ethanol consumption by rats that received bilateral microinjections of a viral vector producing 5-HT(1B) overexpression (HA1B/GFP). Other groups received either control (GFP-only) herpes simplex viral vectors into the medial NAcc shell or were handled briefly with no surgery. All animals were housed singly and had continuous access to water, 6% ethanol, and 12% ethanol in their home cages both before and after surgery. There were no differences in the amount or rate of weight gain, amount of food eaten, or total fluid consumed. There were also no differences in the amount of ethanol consumed between groups prior to surgery. However, after surgery, the HA1B/GFP group consumed twice as much ethanol as the other groups. The main effect of total ethanol consumption was significant (p<.05); the control groups did not differ from each other. Whereas there were no between-group differences in 6% ethanol consumption, there was a large increase in the amount of 12% ethanol consumed by the HA1B/GFP-expressing animals compared to the two control groups as well as to their own presurgery intake (p<.05). We hypothesize that increased 5-HT(1B) expression in NAcc led to either greater reward or reduced aversive effects from the 12% ethanol, thereby leading to increased voluntary ethanol consumption.

Alcohol Drinking↗
PubMed2001

Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts.

Transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. Most non-viral vectors are known to internalize in the cells by endocytosis. Therefore, low transfection efficiency of non-viral vectors may be due to intracellular degradation of input DNA in the endosomes and/or lysosomes. DNA degradation can be inhibited either by inactivating the lysosomal enzymes or obliterating endosome fusion to lysosomes using lysosomotropic agents. We report here the effects of individual lysosomotropic agents such as chloroquine, polyvinylpyrolidone (PVP) and sucrose on beta-gal expression in cultured fibroblasts COS, 293 and CHO. Cell viability was influenced by type, exposure time and concentration of lysosomotropic agents. Exposure to chloroquine at high concentration (1000 microM) or more than 4 h at any concentration (10-1000 microM) caused extensive cell death, however, cytotoxicity due to sucrose (5-500 mM) and PVP (0.01-1 mg/ml) was minimal in the cell lines tested. All the agents utilized in this study enhanced the gene expression and the transfection efficiency followed the order of sucrose>chloroquine>PVP at the concentrations used in all cell lines. Results suggest that lysosomotropic agents can enhance transfection efficiency but the degree of transgene expression may be cell- and agent-specific. Of the agents studied, sucrose appears to be an attractive agent in improving gene expression without toxic effect in the cultured fibroblasts. Thus, it can be used as an excipient in the formulation of new gene delivery systems.

Animals↗
PubMed2006

Gene transfer of soluble transforming growth factor type II receptor by in vivo electroporation attenuates lung injury and fibrosis.

BACKGROUND: Transforming growth factor-beta1 (TGF-beta1) has the potential to induce acute inflammation and apoptosis in lung epithelial cells and plays a central role in subsequent fibrosis. AIMS: To examine a new anti-TGF-beta1 therapy against lung injury and fibrosis, which comprises the transfection of soluble TGF type II receptor (sTGFRII) gene into skeletal muscles by in vivo electroporation. METHODS: Soluble TGFRII was detectable between 1 and 14 days in the serum and significantly increased between 3 and 10 days after gene transfer into muscles. Based on these findings, the sTGFRII gene was injected at 3 days before or 4 days after the bleomycin instillation in order to examine the significance of TGF-beta1 on the early inflammatory phase (day 0 to day 7) or the fibrotic phase (day 7 to day 14) in this model. RESULTS: Transfection of sTGFRII gene at 3 days before or 4 days after bleomycin instillation significantly attenuated apoptosis, injury, and fibrosis at 7 or 14 days, respectively. This method does not require the use of viral vector or neutralising antibody, and it is therefore possible to avoid problems regarding the pathogenicity of the viral vector or immunocomplex. CONCLUSIONS: This novel anti-TGF-beta1 strategy may have clinical application in the treatment of lung injury and fibrosis.

Animals↗
PubMed1997

Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system.

Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less. This activity was approximately 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.

Animals↗
PubMed2005

Ex vivo transduction of human dermal tissue structures for autologous implantation production and delivery of therapeutic proteins.

Systemic delivery of therapeutic proteins through gene transfer approaches has been carried out mostly by ex vivo transduction of single cells or by direct in vivo injection of an expression vector. In this work an intact miniature biopsy of human dermis (microdermis) is harvested and transduced ex vivo by a viral vector encoding a gene for the therapeutic protein. The microdermis preserves its three-dimensional structure and viability during the ex vivo manipulations. Furthermore, upon transduction with adenoviral and adeno-associated viral vectors the microdermis secretes recombinant human erythropoietin (hEPO). Biochemical analysis of the secreted hEPO showed similarity to the clinically approved recombinant hEPO. Subcutaneous implantation of microdermal hEPO into SCID mice exhibited hEPO secretion in the blood circulation and preserved elevated hematocrit for several months, demonstrating the technology's potential for sustained delivery of protein therapeutics.

Adenoviridae↗
PubMed1996

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.

Animals↗
PubMed2002

Characterisation of LMD virus-like nanoparticles self-assembled from cationic liposomes, adenovirus core peptide mu and plasmid DNA.

Liposome:mu:DNA (LMD) is a ternary nucleic acid delivery system built around the mu peptide associated with the condensed core complex of the adenovirus. LMD is prepared by precondensing plasmid DNA (D) with mu peptide (M) in a 1:0.6 (w/w) ratio and then combining these mu:DNA (MD) complexes with extruded cationic liposomes (L) resulting in a final lipid:mu:DNA ratio of 12:0.6:1 (w/w/w). Correct buffer conditions, reagent concentrations and rates of mixing are all crucial to success. However, once optimal conditions are established, homogeneous LMD particles (120 +/- 30 nm) will result that each appear to comprise an MD particle encapsulated within a cationic bilammellar liposome. LMD particles can be formulated reproducibly, they are amenable to long-term storage (>1 month) at -80 degrees C and are stable to aggregation at a plasmid DNA concentration up to 5 mg/ml (15 mM nucleotide concentration). Furthermore, LMD transfections are significantly more time and dose efficient in vitro than cationic liposome-plasmid DNA (LD) transfections. Transfection times as short as 10 min and plasmid DNA doses as low as 0.001 microg/well result in significant gene expression. LMD transfections will also take place in the presence of biological fluids (eg up to 100% serum) giving 15-25% the level of gene expression observed in the absence of serum. Results from confocal microscopy experiments using fluorescent-labelled LMD particles suggest that endocytosis is not a significant barrier to LMD transfection, although the nuclear membrane still is. We also confirm that topical lung transfection in vivo by LMD is at least equal in absolute terms with transfection mediated by GL-67:DOPE:DMPE-PEG(5000) (1:2:0.05 m/m/m), an accepted 'gold-standard' non-viral vector system for topical lung transfection, and is in fact at least six-fold more dose efficient. All these features make LMD an important new non-viral vector platform system from which to derive tailor-made non-viral delivery systems by a process of systematic modular upgrading.

3T3 Cells↗