Reduced Heterochromatin Formation on the pFAR4 Miniplasmid Allows Sustained Transgene Expression in the Mouse Liver.

Non-viral gene delivery into the liver generally mediates a transient transgene expression. A comparative analysis was performed using two gene vectors, pFAR4 and pKAR4, which differ by the absence or presence of an antibiotic resistance marker, respectively. Both plasmids carried the same eukaryotic expression cassette composed of a sulfamidase (Sgsh) cDNA expressed from the human alpha antitrypsin liver-specific promoter. Hydrodynamic injection of the pFAR4 construct resulted in prolonged sulfamidase secretion from the liver, whereas delivery of the pKAR4 construct led to a sharp decrease in circulating enzyme. After induction of hepatocyte division, a rapid decline of sulfamidase expression occurred, indicating that the pFAR4 derivative was mostly episomal. Quantification analyses revealed that both plasmids were present at similar copy numbers, whereas Sgsh transcript levels remained high only in mice infused with the pFAR4 construct. Using a chromatin immunoprecipitation assay, it was established that the 5' end of the expression cassette carried by pKAR4 exhibited a 7.9-fold higher heterochromatin-to-euchromatin ratio than the pFAR4 construct, whereas a bisulfite treatment did not highlight any obvious differences in the methylation status of the two plasmids. Thus, by preventing transgene expression silencing, the pFAR4 gene vector allows a sustained transgene product secretion from the liver.

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells.

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

Improved molecular platform for the gene therapy of rare diseases by liver protein secretion.

Many rare monogenic diseases are treated by protein replacement therapy, in which the missing protein is repetitively administered to the patient. However, in several cases, the missing protein is required at a high and sustained level, which renders protein therapy far from being adequate. As an alternative, a gene therapy treatment ensuring a sustained effectiveness would be particularly valuable. Liver is an optimal organ for the secretion and systemic distribution of a therapeutic transgene product. Cutting edge non-viral gene therapy tools were tested in order to produce a high and sustained level of therapeutic protein secretion by the liver using the hydrodynamic delivery technique. The use of S/MAR matrix attachment region provided a slight, however not statistically significant, increase in the expression of a reporter gene in the liver. We have selected the von Willebrand Factor (vWF) gene as a particularly challenging large gene (8.4 kb) for liver delivery and expression, and also because a high vWF blood concentration is required for disease correction. By using the optimized miniplasmid pFAR free of antibiotic resistance gene together with the Sleeping Beauty transposon and the hyperactive SB100X transposase, we have obtained a sustainable level of vWFblood secretion by the liver, at 65% of physiological level. Our results point to the general use of this plasmid platform using the liver as a protein factory to treat numerous rare disorders by gene therapy.

Electroporation-mediated genetic vaccination for antigen mapping: application to Plasmodium falciparum VAR2CSA protein.

Genetic vaccination, consisting in delivering a genetically engineered plasmid DNA by a non-viral vector or technique into a tissue, is currently of great interest. New delivery technique including DNA transfer by electroporation recently greatly improved the potency of this concept. Because it avoids the step of producing a recombinant protein, it is particularly of use in studying the immunogenic properties of large proteins. Here we describe the use of electroporation mediated DNA immunization to identify important protective epitopes from the large VAR2CSA protein from Plasmodium falciparum implicated in the pathology of placental malaria. Immunizing mice and rabbit with DNA plasmids encoding different fragments of VAR2CSA leads to high titer antisera. Moreover an N-terminal region of the protein was found to induce protective functional antibodies.

The NTS-DBL2X region of VAR2CSA induces cross-reactive antibodies that inhibit adhesion of several Plasmodium falciparum isolates to chondroitin sulfate A.

BACKGROUND: Binding to chondroitin sulfate A by VAR2CSA, a parasite protein expressed on infected erythrocytes, allows placental sequestration of Plasmodium falciparum-infected erythrocytes. This leads to severe consequences such as maternal anemia, stillbirths, and intrauterine growth retardation. The latter has been clearly associated to increased morbidity and mortality of the infants. Acquired anti-VAR2CSA antibodies have been associated with improved pregnancy outcomes, suggesting a vaccine could prevent the syndrome. However, identifying functionally important regions in the large VAR2CSA protein is difficult. METHODS: Using genetic immunization, we raised polyclonal antisera against overlapping segments of VAR2CSA in mice and rabbits. The adhesion-inhibition capacities of induced antisera and of specific antibodies purified from plasma of malaria-exposed pregnant women were assessed on laboratory-adapted parasite lines and field isolates expressing VAR2CSA. Competition enzyme-linked immunosorbent assay (ELISA) was employed to analyze functional resemblance between antibodies induced in animals and those naturally acquired by immune multigravidae. RESULTS: Antibodies targeting the N-terminal sequence (NTS) up to DBL2X (NTS-DBL2X) efficiently blocked parasite adhesion to chondroitin sulfate A in a manner similar to that of antibodies raised against the entire VAR2CSA extracellular domain. Interestingly, naturally acquired antibodies and those induced by vaccination against NTS-DBL2X target overlapping strain-transcendent anti-adhesion epitopes. CONCLUSIONS: This study highlights an important step achieved toward development of a protective vaccine against placental malaria.

Insight into antigenic diversity of VAR2CSA-DBL5ε domain from multiple Plasmodium falciparum placental isolates.

BACKGROUND: Protection against pregnancy associated malaria (PAM) is associated with high levels of anti-VAR2CSA antibodies. This protection is obtained by the parity dependent acquisition of anti-VAR2CSA antibodies. Distinct parity-associated molecular signatures have been identified in VAR2CSA domains. These two observations combined point to the importance of identifying VAR2CSA sequence variation, which facilitate parasitic evasion or subversion of host immune response. Highly conserved domains of VAR2CSA such as DBL5ε are likely to contain conserved epitopes, and therefore do constitute attractive targets for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: VAR2CSA DBL5ε-domain sequences obtained from cDNA of 40 placental isolates were analysed by a combination of experimental and in silico methods. Competition ELISA assays on two DBL5ε variants, using plasma samples from women from two different areas and specific mice hyperimmune plasma, indicated that DBL5ε possess conserved and cross-reactive B cell epitopes. Peptide ELISA identified conserved areas that are recognised by naturally acquired antibodies. Specific antibodies against these peptides labelled the native proteins on the surface of placental parasites. Despite high DBL5ε sequence homology among parasite isolates, sequence analyses identified motifs in DBL5ε that discriminate parasites according to donor's parity. Moreover, recombinant proteins of two VAR2CSA DBL5ε variants displayed diverse recognition patterns by plasma from malaria-exposed women, and diverse proteoglycan binding abilities. CONCLUSIONS/SIGNIFICANCE: This study provides insights into conserved and exposed B cell epitopes in DBL5ε that might be a focus for cross reactivity. The importance of sequence variation in VAR2CSA as a critical challenge for vaccine development is highlighted. VAR2CSA conformation seems to be essential to its functionality. Therefore, identification of sequence variation sites in distinct locations within VAR2CSA, affecting antigenicity and/or binding properties, is critical to the effort of developing an efficient VAR2CSA-based vaccine. Motifs associated with parasite segregation according to parity constitute one such site.

pFARs, plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cells.

BACKGROUND: Nonviral gene therapy requires a high yield and a low cost production of eukaryotic expression vectors that meet defined criteria such as biosafety and quality of pharmaceutical grade. To fulfil these objectives, we designed a novel antibiotic-free selection system. METHODS: The proposed strategy relies on the suppression of a chromosomal amber mutation by a plasmid-borne function. We first introduced a nonsense mutation into the essential Escherichia coli thyA gene, resulting in thymidine auxotrophy. The bacterial strain was optimized for the production of small and novel plasmids free of antibiotic resistance markers (pFARs) and encoding an amber suppressor t-RNA. Finally, the potentiality of pFARs as eukaryotic expression vectors was assessed by monitoring luciferase activities after electrotransfer of LUC-encoding plasmids into various tissues. RESULTS: The introduction of pFARs into the optimized bacterial strain restored normal growth to the auxotrophic mutant and allowed an efficient production of monomeric supercoiled plasmids. The electrotransfer of LUC-encoding pFAR into muscle led to high luciferase activities, demonstrating an efficient gene delivery. In transplanted tumours, transgene expression levels were superior after electrotransfer of the pFAR derivative compared to a plasmid carrying a kanamycin resistance gene. Finally, in skin, whereas luciferase activities decreased within 3 weeks after intradermal electrotransfer of a conventional expression vector, sustained luciferase expression was observed with the pFAR plasmid. CONCLUSIONS: Thus, we have designed a novel strategy for the efficient production of biosafe plasmids and demonstrated their potentiality for nonviral gene delivery and high-level transgene expression in several tissues.