Meganuclease-mediated Inhibition of HSV1 Infection in Cultured Cells.

Herpes simplex virus type 1 (HSV1) is a major health problem. As for most viral diseases, current antiviral treatments are based on the inhibition of viral replication once it has already started. As a consequence, they impair neither the viral cycle at its early stages nor the latent form of the virus, and thus cannot be considered as real preventive treatments. Latent HSV1 virus could be addressed by rare cutting endonucleases, such as meganucleases. With the aim of a proof of concept study, we generated several meganucleases recognizing HSV1 sequences, and assessed their antiviral activity in cultured cells. We demonstrate that expression of these proteins in African green monkey kidney fibroblast (COS-7) and BSR cells inhibits infection by HSV1, at low and moderate multiplicities of infection (MOIs), inducing a significant reduction of the viral load. Furthermore, the remaining viral genomes display a high rate of mutation (up to 16%) at the meganuclease cleavage site, consistent with a mechanism of action based on the cleavage of the viral genome. This specific mechanism of action qualifies meganucleases as an alternative class of antiviral agent, with the potential to address replicative as well as latent DNA viral forms.

Integrin-targeted nanocomplexes for tumour specific delivery and therapy by systemic administration.

Nanoparticle formulations offer opportunities for tumour delivery of therapeutic reagents. The Receptor-Targeted Nanocomplex (RTN) formulation consists of a PEGylated, endosomally-cleavable lipid and an RGD integrin-targeting, endosomally-cleavable peptide. Nancomplexes self-assemble on mixing with plasmid DNA to produce nanoparticles of about 100 nm. The environmentally-sensitive linkers promote intracellular disassembly and release of the DNA. RTNs carrying luciferase genes were administered intravenously to mice carrying subcutaneous neuroblastoma tumours. Luciferase expression was much higher in tumours than in liver, spleen and lungs while plasmid biodistribution studies supported the expression data. Transfection in tumours was enhanced two-fold by integrin-targeting peptides compared to non-targeted nanocomplexes. RTNs containing the interleukin-2 (IL-2) and IL-12 genes were administered intravenously with seven doses at 48 h intervals and tumour growth monitored. Tumours from treated animals were approximately 75% smaller on day 11 compared with RTNs containing control plasmids with one third of treated mice surviving long-term. Extensive leukocyte infiltration, decreased vascularization and increased necrotic areas were observed in the tumours from IL2/IL12 treated animals. Splenocytes from re-challenged mice displayed enhanced IL-2 production following Neuro-2A co-culture, which, combined with infiltration studies, suggested a cytotoxic T cell-mediated9 tumour-rejection process. The integrin-targeted RTN formulation may have broader applications in the further development of cancer therapeutics.

Tumor-specific gene transfer with receptor-mediated nanocomplexes modified by polyethylene glycol shielding and endosomally cleavable lipid and peptide linkers.

Synthetic nanoparticle formulations have the potential for tumor-targeted gene delivery. Receptor-targeted nanocomplex (RTN) formulations comprise mixtures of cationic liposomes and targeting peptides that self-assemble on mixing with nucleic acids. RTN formulations were prepared containing different polyethylene glycol (PEG)ylated lipids with esterase-cleavable linkers (e.g., ME42) to promote intracellular PEG detachment and nanoparticle disassembly. In addition, integrin-targeting peptides (peptide ME27) were tested with endosomal furin- and cathepsin B-cleavable peptide linkers located between the integrin-binding ligand and the K(16) nucleic acid-binding domain to promote intracellular disengagement from the receptor. ME42/ME27 RTNs formed stable particles of <200 nm in isotonic salt buffers, compared with 4-microm particles formed by un-PEGylated RTNs. Transfection efficiency by PEG-modified, cleavable RTNs improved approximately 2-fold in 4 different cell lines, with 80% efficiency in murine neuroblastoma cells. In an in vivo model of neuroblastoma, ME42/ME27 RTNs delivering luciferase genes were tumor specific, with little expression in other organs tested. PEGylation of the RTNs enhanced luciferase transfection 5-fold over non-PEG formulations, whereas the cleavability of the peptide ME27 enhanced transfection 4-fold over that of RTNs with noncleavable peptides. Cleavability of the lipid for in vivo transfections had no effect. PEGylated, cleavable RTN formulations offer prospects for tumor-specific therapeutic gene transfer.

Stabilized integrin-targeting ternary LPD (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell.

Recent research in the field of nonviral gene delivery vectors has focused on preparing nanoparticles that are stabilized by the incorporation of a PEG coating and where one of the vector components is also cleavable. Here,we describe the synthesis, formulation, transfection properties, and biophysical studies of a PEG-stabilized ternary lipopolyplex vector in which, for the first time, both the lipid and peptide components are designed to be cleaved once the vector has been internalized. A series of cationic lipids, bearing short tri- or hexaethylene glycol groups, attached to the headgroup via an ester linkage, has been prepared. Trifunctional peptides have also been prepared, consisting of a Lys(16) sequence at the N-terminus (to bind and condense plasmid DNA); a spacer group (containing a sequence recognized and cleaved by endosomal enzymes) and an optional PEG4 amino acid; and an integrin-targeting cyclic peptide sequence (allowing the resulting nanoparticle to be internalized via receptor-mediated endocytosis). Differing combinations of these lipids and peptides have been formulated with DOPE and with plasmid DNA, and complex stability, transfection, and cleavage studies carried out. It was shown that optimal transfection activities in a range of cell types and complex stabilities were achieved with lipids bearing short cleavable triethylene glycol moieties, whereas the incorporation of PEG4 amino acids into the cleavable peptides had little effect. We have synthesized appropriate fluorescently labeled components and have studied the uptake of the vector, endosomal escape, peptide cleavage, and plasmid transport to the nucleus in breast cancer cells using confocal microscopy. We have also studied the morphology of these compact, stabilized vectors using cryo-EM.

In vivo gene delivery in the mouse lung with lactosylated polyethylenimine, questioning the relevance of in vitro experiments.

Polyethylenimine (PEI) is an efficient vector for in vitro and in vivo gene transfer into respiratory cells. Glycosylated PEIs were shown to enhance in vitro gene transfer by favoring the complex entry into the airway cells. The aim of our study was to evaluate the in vivo efficiency of gene transfer mediated by glycosylated PEIs in the mouse lung and to determine the transfected cell type and the intracellular trafficking of the complexes. Upon nasal instillation in mice of complexes made with various glycosylated PEIs, a high luciferase activity was observed while the green fluorescent protein (GFP) expression was similar for all the vectors tested with few cells expressing GFP. Complexes made with lactosylated PEI were then labeled and their localization studied by confocal microscopy. In the lungs, large numbers of complexes were taken up by epithelial cell which were mostly alveolar cells. In the airways, complex uptake varied greatly, depending on the area observed. Eight hours upon nasal instillation and in contrast with the in vitro situation, a dissociation between the plasmid DNA and the lactosylated PEI was usually observed, leading to the plasmid mostly localized in lysosomes and the Lac-PEI localized in the nucleus. These results emphasize the need to engineer a plasmid able by itself to overcome the nuclear barrier and to quickly move to in vivo experiments to select the best carrier.

Acid cleavable PEG-lipids for applications in a ternary gene delivery vector.

A novel class of pH-sensitive PEG lipids bearing acid-cleavable acetal linkages and short PEG chains have been synthesised and used in ternary vector formulations. The cleavage pH was influenced by structural components including the terminal PEG moiety and spacer length.

Cytoskeletal involvement in the cellular trafficking of plasmid/PEI derivative complexes.

We have studied the cytoskeletal involvement in the cellular trafficking of complexes made with plasmid/PEI or plasmid/lactosylated PEI in cystic fibrosis airway epithelial cells (SigmaCFTE29o- cells). Complexes were incubated in the presence of cytoskeletal inhibitors, and the number of transfected cells was determined by flow cytometry. Complexes were also generated with fluorescein-labeled PEI derivatives and the cell fluorescence intensity was determined by flow cytometry. In the presence of cytochalasin D to depolymerize actin filaments or nocodazole to disrupt microtubules, gene transfer efficiency with both PEI derivatives was decreased by 90%. The uptake of fluoresceinylated complexes studied by flow cytometry was decreased by 50% in the presence of cytochalasin D for both types of complexes (p<0.005) and unchanged in the presence of nocodazole. When cytoskeletal inhibitors were added to the cell culture after the complex uptake had occurred, gene transfer efficiency was decreased by 75% and 50% in the presence of nocodazole and cytochalasin D, respectively. Upon nocodazole-microtubule network disruption, the lysosomal localization of complexes was reduced, as assessed by confocal microscopy. Our results show a major cytoskeletal involvement in the cellular trafficking of complexes made with both PEI derivatives: actin filaments mainly in complex uptake, and microtubules in the trafficking of complexes towards the nucleus, probably through guided transport of complex-containing endosomal vesicles.

Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives?

BACKGROUND: To investigate the nuclear import mechanism of plasmid/polyethylenimine (PEI) derivative complexes and the putative nuclear targeting of therapeutic genes by the use of oligosaccharides, we have studied the nuclear import of plasmid DNA complexed either with PEI or with lactosylated PEI (Lac-PEI) in cystic fibrosis human airway epithelial cells ( summation operatorCFTE29o- cells). METHODS AND RESULTS: Cells were synchronized by a double-thymidine block protocol and gene transfer efficiency was evaluated: Lac-PEI- and PEI-mediated gene transfer was greatly increased when cells have undergone mitosis during the course of transfection. However, both types of complexes were able to transfect some growth-arrested cells. When the nuclear import of plasmid/Lac-PEI or plasmid/unsubstituted PEI complexes was studied in digitonin-permeabilized cells, the nuclear uptake of both types of complexes did not follow the classic pathway of nuclear localization sequence (NLS)-containing proteins and lactose residues did not act as a nuclear localization signal. CONCLUSIONS: Our results show that for complexes made with PEI derivatives, the major route for plasmid DNA nuclear entry is a passive nuclear importation during mitosis when the nuclear membrane temporarily breaks down. However, albeit to a lesser extent as that observed in dividing cells, a plasmid DNA importation also occurs in nondividing cells by a yet unknown mechanism.

Transcription of plasmid DNA: influence of plasmid DNA/polyethylenimine complex formation.

Polyethylenimine (PEI) is one of the most potent non-viral vectors. We have developed a lactosylated PEI (Lac-PEI) to enhance cell-specific transfection and have shown that Lac-PEI is more efficient than unsubstituted PEI for gene transfer into immortalized cystic fibrosis airway epithelial SigmaCFTE29o-cells. As both intact PEI/plasmid and Lac-PEI/plasmid complexes are found in the cell nucleus, we have investigated the transcription efficiency of the plasmid complexed with PEI or Lac-PEI, according to the polymer nitrogen/DNA phosphate (N/P) ratio (from 0 to 20). The initiation of transgene transcription was analyzed in an acellular nuclease S1 transcription assay. For both PEI and Lac-PEI complexes, transcription efficiency varied with the N/P ratio of the complexes. Transcription inhibition was observed when plasmid DNA was either loosely (N/P<5) or tightly condensed (N/P>15). For an N/P ratio of 5 and up to 15, transcription of the complexed plasmid was as efficient as that of the free plasmid. Similar results were observed when gene expression was studied after nuclear microinjection of the complexes into SigmaCFTE29o-cells. Our study shows that condensation of DNA influences the accessibility of the plasmid to the transcription machinery. Interestingly, the charge ratios that allow the most efficient transcription are those usually known to be the most efficient for gene transfer in vitro and in vivo.

Potocytosis and cellular exit of complexes as cellular pathways for gene delivery by polycations.

BACKGROUND: Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified. METHODS AND RESULTS: The trafficking of plasmid DNA complexed to glycosylated polylysines or polyethylenimine (PEI) derivatives was studied by electron microscopy of human airway epithelial cells. The cellular processing of complexes varied with their size and the polycation derivative used: large complexes (> 200 nm) made with all polycationic vectors studied were internalized by macropinocytosis. In contrast, intermediate (100-200 nm) ligand-coupled polylysine and PEI complexes primarily entered through clathrin-coated pits. Complexes were then found in endosomal vesicles, accumulated in lysosomes or vesicles near the nucleus and their nuclear entry was limited. For the population of small complexes (< or = 100 nm) obtained with PEI derivatives, they were internalized through caveolae and pursued a traffic pattern of potocytosis to the endoplasmic reticulum where their fate remains unclear. Finally, some complexes exited the cells either by regurgitation when PEI derivatives were used or through an exosome-like pathway for glycosylated-polylysine complexes. CONCLUSIONS: The different pathways of complex trafficking observed in relation with complex size imply the development and study of vectors forming complexes with definite size. Moreover, the complex exit we describe may contribute to the well-established short-term efficiency of gene transfer based on synthetic vectors. It favors the engineering of vectors allowing repeated treatment.

Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexes.

BACKGROUND: As we have previously shown that lactosylated polyethylenimine (PEI) is the most efficient glycosylated PEI for gene transfer into human airway epithelial cells in primary culture, we have studied here the role of the lactose residue in the enhancement of gene transfer efficiency observed with lactosylated PEI as compared with unsubstituted PEI in immortalized (Sigma CFTE29o- cells) and primary human airway epithelial cells. METHODS AND RESULTS: After three transfections of 1 h performed daily, 60% of Sigma CFTE29o- cells were transfected with lactosylated PEI, whereas 25% of cells were transfected with unsubstituted PEI (p < 0.05). Cell viability was 1.8-fold greater with lactosylated PEI as compared with unsubstituted PEI (p < 0.05). As assessed by flow cytometry, the cellular uptake of lactosylated complexes was greater than that of complexes made with unsubstituted PEI (p < 0.05) and involved mostly a receptor-mediated endocytosis. The study of the intracellular trafficking in airway epithelial cells of complexes showed an endosomal and lysosomal accumulation of lactosylated complexes. In the presence of a proton pump inhibitor, the level of lactosylated and unsubstituted PEI-mediated gene expression was reduced more than 20-fold, whereas the cell viability increased to almost 100%. For both complexes, a nuclear localization was observed for less than 5% of intracellular complexes. CONCLUSIONS: Our results show that the greater gene transfer efficiency observed for lactosylated complexes may be attributed to a higher amount of lactosylated complexes incorporated by airway epithelial cells and a lower cytotoxicity that might be related to reduced endosomolytic properties. However, the lactose residues substituting the PEI did not promote the entry of the plasmid into the nucleus.