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Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA.

ABSTRACT: Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA-virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA-virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA-virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA-virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

SUBMITTER: de Jonge J

PROVIDER: S-EPMC1925238 | biostudies-other | 2007 Jul

REPOSITORIES: biostudies-other

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Project description:UNLABELLED:Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. IMPORTANCE:Diseases caused by multidrug-resistant bacteria are taking an important toll with respect to human morbidity and mortality. The most relevant antibiotic resistance genes come to human pathogens carried by plasmids, mainly using conjugation as a transmission mechanism. Here, we identified and characterized a series of compounds that were active against several plasmid groups of clinical relevance, in a wide variety of bacterial hosts. These inhibitors might be used for fighting antibiotic-resistance dissemination by inhibiting conjugation. Potential inhibitors could be used in specific settings (e.g., farm, fish factory, or even clinical settings) to investigate their effect in the eradication of undesired resistances.

Project description:Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines by Mycobacterium smegmatis or M. bovis BCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer using M. smegmatis or BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant-albeit only 1.7-fold-increase in the level of plasmid transfer to eukaryotic cells infected with M. smegmatis hyperconjugation mutants. M. smegmatis strains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation in oriM resulted in a threefold increase in gene transfer to J774 cells. Moreover, M. smegmatis strains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120(h)(E)) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P < 0.05) and threefold (P < 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120(h)(E). M. smegmatis and other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

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Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA.

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