pFAR plasmids: New Eukaryotic Expression Vectors for Gene Therapy, devoid of Antibiotic Resistance Markers

Efficient production of eukaryotic expression vectors requires the selection of plasmid-containing bacteria. To avoid the risk of dissemination of antibiotic resistance markers, we developed a new system to produce a family of plasmids Free of Antibiotic Resistance genes, called pFARs. The strategy is based on the suppression of a chromosomal nonsense mutation by a plasmid-borne function. The amber mutation was introduced into the Escherichia coli thyA gene that encodes a thymidylate synthase required for dTMP synthesis, resulting in thymidine auxotrophy. In parallel, a small plasmid vector that carries an amber suppressor t-RNA gene was entirely synthesised. The introduction of pFAR plasmids into an optimised thyA mutant restored normal growth to the auxotrophic strain, and led to an efficient production of monomeric supercoiled plasmids, as required for clinical trials. Luciferase activities measured after intramuscular injection and electrotransfer of LUC-encoding pFAR vector were similar to those obtained with a commercial vector containing the same expression cassette. Interestingly, whereas luciferase activities decreased within three weeks after intradermal electrotransfer of conventional expression vectors, sustained levels were observed with the pFAR derivative. Thus, pFAR plasmids represent a novel family of biosafe eukaryotic expression vectors, suitable for gene therapy

Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties

BACKGROUND: Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. RESULTS: We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. CONCLUSIONS: The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer

Careful adjustment of Epo non-viral gene therapy for β-thalassemic anaemia treatment

BACKGROUND: In situ production of a secreted therapeutic protein is one of the major gene therapy applications. Nevertheless, the plasmatic secretion peak of transgenic protein may be deleterious in many gene therapy applications including Epo gene therapy. Epo gene transfer appears to be a promising alternative to recombinant Epo therapy for severe anaemia treatment despite polycythemia was reached in many previous studies. Therefore, an accurate level of transgene expression is required for Epo application safety. The aim of this study was to adapt posology and administration schedule of a chosen therapeutic gene to avoid this potentially toxic plasmatic peak and maintain treatment efficiency. The therapeutic potential of repeated muscular electrotransfer of light Epo-plasmid doses was evaluated for anaemia treatment in β-thalassemic mice. METHODS: Muscular electrotransfer of 1 μg, 1.5 μg, 2 μg 4 μg or 6 μg of Epo-plasmid was performed in β-thalassemic mice. Electrotransfer was repeated first after 3.5 or 5 weeks first as a initiating dose and then according to hematocrit evolution. RESULTS: Muscular electrotransfer of the 1.5 μg Epo-plasmid dose repeated first after 5 weeks and then every 3 months was sufficient to restore a subnormal hematrocrit in β-thalassemic mice for more than 9 months. CONCLUSION: This strategy led to efficient, long-lasting and non-toxic treatment of β-thalassemic mouse anaemia avoiding the deleterious initial hematocrit peak and maintaining a normal hematocrit with small fluctuation amplitude. This repeat delivery protocol of light doses of therapeutic gene could be applied to a wide variety of candidate genes as it leads to therapeutic effect reiterations and increases safety by allowing careful therapeutic adjustments

Host-Enhanced Phenyl-Perfluorophenyl Polar-π Interactions.

Phenyl-perfluorophenyl polar-π interactions have been revisited for the design and fabrication of functional supramolecular systems. The relatively weak associative interactions (ΔG ≈ -1.0 kcal/mol) have limited their use in aqueous self-assembly to date. Herein, we propose a strategy to strengthen phenyl-perfluorophenyl polar-π interactions by encapsulation within a synthetic host, thus increasing the binding affinity to ΔG= -15.5 kcal/mol upon formation of heteroternary complexes through social self-sorting. These heteroternary complexes were used as dynamic, yet strong, cross-linkers in the fabrication of supramolecular gels, which exhibited excellent viscoelasticity, stretchability, self-recovery, self-healing, and energy dissipation. This work unveils a general approach to exploit host-enhanced polar-π interactions in the design of robust aqueous supramolecular systems

In vivo optical imaging with rare earth doped Ca₂Si₅N₈ persistent luminescence nanoparticles

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Observing Single Molecules Complexing with Cucurbit[7]uril through Nanogap Surface-Enhanced Raman Spectroscopy.

In recent years, single-molecule sensitivity achievable by surface-enhanced Raman spectroscopy (SERS) has been widely reported. We use this to investigate supramolecular host-guest chemistry with the macrocyclic host cucurbit[7]uril, on a few-to-single-molecule level. A nanogap geometry, comprising individual gold nanoparticles on a planar gold surface spaced by a single layer of molecules, gives intense SERS signals. Plasmonic coupling between the particle and the surface leads to strongly enhanced optical fields in the gap between them, with single-molecule sensitivity established using a modification of the well-known bianalyte method. Changes in the Raman modes of the host molecule are observed when single guests included inside its cavity internally stretch it. Anisotropic intermolecular interactions with the guest are found which show additional distinct features in the Raman modes of the host molecule.The authors acknowledge funding from Walters-Kundert Trust, EPSRC (EP/K028510/1, EP/G060649/1, EP/ H007024/1, ERC LINASS 320503), an ERC starting investigator grant (ASPiRe 240629), EU CUBiHOLE grant and the Defence Science and Technology Laboratory (DSTL). S.K. thanks Krebs Memorial Scholarship (The Biochemical Society) and Cambridge Commonwealth Trust for funding.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acs.jpclett.5b0253