Examination of the effect of increasing the number of intra-disulfide amino functional groups on the performance of small molecule cyclic polyamine disulfide vectors.

Establishing structure-activity relationships is vital if the efficacy of non-viral vectors is to match that of their viral counter-parts. Recently, we reported on the ability of a series of small molecule, cyclic polyamine disulfides to condense and cage plasmid DNA (pDNA) by a process of thermodynamically controlled templated polymerization, leading to a series of corresponding pDNA-polyplex nanoparticles able to mediate high levels of transfection with no associated cytotoxicities. The leading cyclic polyamine disulfide was shown to be the spermine tetra-amine disulfide (TetraN-3,4,3). Herein we report on the significantly more challenging syntheses of cyclic disulfides with longer polyamine motifs. Two new cyclic polyamine disulfides, based on hexa- and octa-amine inserts, were prepared and their transfection efficacies and cytotoxicities compared with our previously reported cyclic tri- and tetra-amine disulfides. The new cyclic hexa- and octa-amine disulfides prove more effective at transfection in vitro, especially of lung epithelial A549 cell line. By contrast, our original cyclic tetra-amine disulfide remains the most efficient agent for the transfection of lung epithelial cells in vivo following intra-nasal administration. Hypothetical mechanistic reasons are presented to explain this outcome. Our data in toto support the concept of shorter cyclic polyamine disulfides as preferred agents for polycation-mediated controlled condensation and functional delivery of pDNA to lung epithelial cells in vivo.

Efficient topical delivery of plasmid DNA to lung in vivo mediated by putative triggered, PEGylated pDNA nanoparticles.

Non-viral vectors are considered safer than viral vectors and show clinical potential, but remain less efficient in terms of DNA delivery. Here we report how cationic liposomes, prepared from new cationic lipid, N',N',-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide (DODAG) and neutral lipid dioleoyl-L-α-phos-phatidylethanolamine (DOPE), can be formulated with plasmid DNA (pDNA) in the presence of stabilizer cholesteryl-oxycarbonylpolyethlylene glycol(4600) (PEG(4600)-Chol) giving PEGylated pDNA nanoparticles (pDNA-ABC nanoparticles) that are proposed to be half-life triggered nanoparticles. In particular, the PEGylated pDNA nanoparticle formulation DODAG/DOPE/PEG(4600)-Chol (43:43:14, m/m/m)-pDNA (total lipid/pDNA ratio 4:1 w/w) (pTRANSplus nanoparticles) is shown to mediate efficient transfection of murine lung tissue in vivo. Levels of transfection compare well with the results of polyethylenimine (PEI) mediated pDNA transfection in vivo and even of adenovirus mediated transduction. Cryo-EM imaging indicates that pTRANSplus formulations are somewhat heterogeneous but do consist primarily of bilammellar lipoplex nanoparticles with a few multilammellar nanoparticle aggregates. Lung histology confirms that pTRANSplus mediated transfection in vivo targets substantially the epithelial cells of bronchii and bronchioli airway passages. The pTRANSplus nanoparticle system is a useful new starting point for nucleic acid therapeutic strategies to counter lung disorders such as viral infection and possibly cystic fibrosis.

Bioresponsive small molecule polyamines as noncytotoxic alternative to polyethylenimine.

Nonviral gene therapy continues to require novel synthetic vectors to deliver therapeutic nucleic acids effectively and safely. The majority of synthetic nonviral vectors employed in clinical trials to date have been cationic liposomes; however, cationic polymers are attracting increasing attention. One of the few cationic polymers to enter clinical trials has been polyethylenimine (PEI); however, doubts remain over its cytotoxicity, and in addition it displays lower levels of transfection than viral systems. Herein, we report on the development of a series of small molecule analogues of PEI that are bioresponsive to the presence of pDNA, forming poly(disulfide)s that are capable of efficacious transfection with no associated toxicity. The most effective small molecule developed, a cyclic disulfide based upon a spermine backbone, is shown to form very well-defined polyplexes (100-200 nm in diameter) that mediate murine lung transfection in vivo to within an order of magnitude of in vivo jetPEI, and at the same time display a much improved cytotoxicity profile.

Combining keratinocyte growth factor transfection into the airways and tracheal occlusion in a fetal sheep model of congenital diaphragmatic hernia.

BACKGROUND: In utero tracheal occlusion (TO) has been developed to improve the lung hypoplasia associated with congenital diaphragmatic hernia (CDH). However, although TO stimulates fetal lung growth, it results in a decrease of alveolar type II cells (ATII) and surfactant production. Because keratinocyte growth factor (KGF) is a potent stimulus of ATII proliferation and maturation, we evaluated, in a fetal lamb model of CDH, a gene therapy strategy combining TO and ovine KGF transfection into the fetal airways using bisguanidinium-tren-cholesterol/dioleoyl-phosphatidylethanolamine (BGTC/DOPE) cationic liposomes. METHODS: Three groups of sheep fetuses with CDH and a group of normal fetuses were studied. The fetuses of the three groups with CDH (KGF, Medium and Hernia groups) underwent surgery at 85 days of gestation to create a diaphragmatic hernia. The KGF and medium group fetuses underwent a second surgery step at day 125 to perform TO associated with injection of the KGF transfection mixture (KGF group) or control medium (Medium group), whereas the fetuses of the Hernia group were left untreated. Normal fetuses were used as a control (Normal group). All fetuses were euthanized at 132 days of gestation and various analytical studies [lung weight, radial alveolar count (RAC), KGF and surfactant protein B (SPB) expression, number of ATII cells] were performed to assess the efficiency of KGF transfection and its effects on fetal lung development. RESULTS: TO was associated with lung hyperplasia and increased RAC in the Medium and KGF groups versus the Hernia group. Expression of KGF was increased in the KGF group compared to all other groups and was associated with an increased synthesis of SPB by alveolar cells and an ectopic synthesis of SPB by bronchiolar cells compared to TO treatment alone. CONCLUSIONS: Thus, BGTC/DOPE liposomes can mediate efficient KGF transfection into the airways in a fetal sheep model of CDH. Furthermore, combining KGF transfection and TO resulted not only (as did TO alone) in the correction of the CDH-associated lung hypoplasia and decreased RAC, but also in increased SPB synthesis, suggesting a better maturation of the re-growing lung (compared to TO alone). Additional studies are required to further explore the therapeutic potential of such a combined strategy; in particular, studies evaluating the lung function of in utero-treated CDH lamb newborns.

Kanamycin A-derived cationic lipids as vectors for gene transfection.

Cationic lipids nowadays constitute a promising alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a new class of lipids based upon the use of aminoglycosides as cationic polar headgroups. The encouraging results obtained with a first cholesterol derivative of kanamycin A prompted us to investigate this family of vectors further, by modulating the constituent structural units of the cationic lipid. For this study, we have investigated the transfection properties of a series of new derivatives based on a kanamycin A scaffold. The results primarily confirm that aminoglycoside-based lipids are efficient vectors for gene transfection both in vitro and in vivo (mouse airways). Furthermore, a combination of transfection and physicochemical data revealed that some modifications of the constitutive subunits of kanamycin A-based vectors were associated with substantial changes in their transfection properties.

Novel cationic lipids incorporating an acid-sensitive acylhydrazone linker: synthesis and transfection properties.

Cationic lipid-mediated gene transfection involves uptake of the lipid/DNA complexes via endocytosis, a cellular pathway characterized by a significant drop in pH. Thus, in the present study, we aimed to explore the impact on transfection efficiency of the inclusion of an acid-sensitive acylhydrazone function in the cationic lipid structure. We synthesized and evaluated the transfection properties of a series of four cationic steroid derivatives characterized by an acylhydrazone linkage connecting a guanidinium-based headgroup to a saturated cholestanone or an unsaturated cholest-4-enone hydrophobic domain. Acid-catalyzed hydrolysis was confirmed for all lipids, its rate being highest for those with a cholestanone moiety. The compound bis-guanidinium bis(2-aminoethyl)amine hydrazone (BGBH)-cholest-4-enone was found to mediate efficient gene transfection into various mammalian cell lines in vitro and into the mouse airways in vivo. In vitro transfection studies with BGBH-cholest-4-enone formulations also showed that incorporation of a degradable acylhydrazone bond led to low cytotoxicity and impacted the intracellular trafficking of the lipoplexes. Thus, our work allowed us to identify a cationic lipid structure with an acid-cleavable acylhydrazone linker capable of mediating efficient gene transfection in vitro and in vivo and it thereby provides a basis for further development of related acid-sensitive gene delivery systems.

Gene transfection into fetal sheep airways in utero using guanidinium-cholesterol cationic lipids.

BACKGROUND: Over the last several years, we have developed a novel class of cationic lipids, cholesterol derivatives characterized by polar head groups with guanidinium functions. We have in particular shown that bis(guanidinium)-tren-cholesterol/dioleoylphosphatidylethanolamine (BGTC/DOPE) cationic liposomes can mediate efficient gene transfection into the mouse airways in vivo via direct intratracheal administration or intranasal instillation. As prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we have explored in the present work the feasibility of BGTC-mediated gene transfection into the respiratory tract of fetal sheep in utero. METHODS: Thus, BGTC/DOPE liposomes were complexed with plasmids expressing the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene and the resulting lipoplexes were administered to fetal sheep at 70 days of gestation via surgical replacement of the airway fluid by the transfection mixture followed by tracheal occlusion. The fetal lungs and tracheas were harvested at 72 h and examined for CAT expression and evidence of toxicity. RESULTS: CAT expression was detected in both lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked plasmid DNA. Immunohistochemical analysis showed that airway epithelial cells and some mesenchymal cells were transfected. Pulmonary histopathology of varied severity was however observed under our transfection conditions and manifested as focal epithelial and mesenchymal lesions. CONCLUSIONS: These results show that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium. They also invite the development of optimized BGTC-based formulations and administration conditions with a view to future prenatal gene transfer experiments involving therapeutic genes.

Circulating regulatory anti-T cell receptor antibodies in patients with myasthenia gravis.

Serum anti-T cell receptor (TCR) Ab's are involved in immune regulation directed against pathogenic T cells in experimental models of autoimmune diseases. Our identification of a dominant T cell population expressing the Vbeta5.1 TCR gene (TCRBV5-1), which is responsible for the production of pathogenic anti-acetylcholine receptor (AChR) autoantibodies in HLA-DR3 patients with early-onset myasthenia gravis (EOMG), prompted us to explore the occurrence, reactivity, and regulatory role of anti-TCR Ab's in EOMG patients and disease controls with clearly defined other autoantibodies. In the absence of prior vaccination against the TCR, EOMG patients had elevated anti-Vbeta5.1 Ab's of the IgG class. This increase was restricted largely to EOMG cases with HLA-DR3 and with less severe disease, and it predicted clinical improvement in follow-up studies. EOMG patient sera containing anti-TCR Ab's bound specifically the native TCR on intact Vbeta5.1-expressing cells and specifically inhibited the proliferation and IFN-gamma production of purified Vbeta5.1-expressing cells to alloantigens in mixed lymphocyte reaction and the proliferation of a Vbeta5.1-expressing T cell clone to an AChR peptide, indicating a regulatory function for these Ab's. This evidence of spontaneously active anti-Vbeta5.1 Ab's in EOMG patients suggests dynamic protective immune regulation directed against the excess of pathogenic Vbeta5.1-expressing T cells. Though not sufficient to prevent a chronic, exacerbated autoimmune process, it might be boosted using a TCR peptide as vaccine.

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo.

BACKGROUND: Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature. METHODS AND RESULTS: We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino-sugar-based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin-cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo. CONCLUSIONS: These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo.

Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example.

Artificial self-assembling systems are currently widely investigated as an alternative approach to recombinant viruses for gene transfection in vitro and in vivo. Cationic lipids are particularly attractive, as a great variety of well-characterized reagents can be synthesized from there. Over the last few years, numerous cationic lipid systems have been developed and shown to be efficient for in vitro transfection. However, although some promising results have been reported in the in vivo setting (even in clinical gene therapy trials in man), the in vivo use of cationic lipid-based systems is still problematic, especially when considering the systemic route of administration. Herein, we summarize our own research on a particular class of cationic lipids, cholesterol derivatives characterized by polar headgroups with guanidinium functions, in order to illustrate the basic principles of and the positive results already obtained by cationic lipid-mediated gene delivery as well as the remaining problems that need to be urgently resolved, particularly as regards the systemic administration. In this forward-looking review, we also discuss the present efforts to develop modular systems for improved in vivo transfection. Indeed, lipid-based vectors offer the possibility to create sophisticated modular gene delivery systems capable of self-assembly via hydrophobic interaction between their components, the role of the different functional elements being to help in overcoming the distinct extracellular and cellular barriers to in vivo gene transfection into the various somatic target tissues.