Current Medicinal Chemistry - Volume 11, Issue 2, 2004

Gene carriers based on lipids or polymers or a combination of these - rather than on engineered viruses - are emerging as among the “hottest technologies” for delivering genes into cells for gene therapy and therapeutics. The topic has received increasing attention having been the subject matter of recent news features in highly visible technical Magazines such as Science and Chemical & Engineering News. Indeed, as described in Table 1 of the article by Ewert et al., nearly one quarter of ongoing gene therapy clinical trials are conducted with non-viral methods including lipids, polymers, and naked DNA. This thematic issue of Current Medicinal Chemistry is devoted to a series of reviews focusing on some of the latest findings in the area of synthetic non-viral gene delivery systems for therapeutic applications. A distinguishing feature of the articles is a significant emphasis on understanding vector-DNA complexes and their interactions with cells at the molecular level rather than in a purely empirical manner by trial and error. The first article in this series by Ewert et al. describes the use of cationic lipids as gene vectors. The article describes how the use of modern technologies, including synthesis of tailored molecules and gene expression assays, synchrotron x-ray diffraction for structure determination, and three-dimensional laser-scanning confocal microscopy imaging of lipid-DNA interactions with cells, enables one to unravel structure-function relations with new insights for enhancing transfection efficacy. To date, the main theoretical work on synthetic vectors has focused on cationic lipids complexed with DNA. The review by May and Ben-Shaul updates the reader on recent advances in modeling the structure and thermodynamic stability of cationic lipid-DNA complexes. Although the majority of laboratories working on non-viral gene delivery systems focus on cationic lipids and polymers because of their natural ability to simultaneously condense therapeutic DNA and to attach to mammalian cells via their negative receptor molecules, the relative higher toxicity of the vectors compared to neutral lipid vectors has led some researchers to focus on other strategies. In their review Roux et al. describe a novel process by which DNA, a negative molecule, can be trapped between uncharged lipid membranes for delivery applications, even though the usual electrostatic attractions between DNA and lipid are absent. Davis et al. introduce an entirely new class of novel water-soluble polymeric materials, cyclodextrin-containing polymers, which are shown to self assemble with DNA as a distinct type of polymer delivery technology. Their review outlines the effect of the molecular structure and the role of hydrophobic and hydrophilic interactions as well as chemical modifications of the end groups and the backbone on the activity of these molecules as drug carriers. The final review by Kisak et al. describes the invention of the vesosome, a synthetic carrier consisting of a set of vesicles contained within an outer lipid membrane. The remarkable structure of the vesosome, which is reminiscent of the eukaryotic cell with inner organelles, allows one to compartmentalize for simultaneous delivery of different drug molecules for a range of functions. The ultimate goal of research and development on virus-free carriers is to develop a science base, which will lead to the design and synthesis of optimal carriers of DNA for gene therapy and disease control. On a final note, it should be mentioned that the recent renaissance in the field is also partly because of the realization of the potential of the delivery technology in transferring large pieces of DNA, such as sections containing over a million base pairs, into cells. Thus, one may envision future applications with synthetic-vectors designed to deliver a cassette of human genes together with their regulatory sequences. Such a feat is simply not in the realm of possibilities with current viral vector strategies.

Cationic liposomes (CLs) are used as gene vectors (carriers) in worldwide human clinical trials of non-viral gene therapy. These lipid-gene complexes have the potential of transferring large pieces of DNA of up to 1 million base-pairs into cells. As our understanding of the mechanisms of action of CL-DNA complexes remains poor, transfection efficiencies are still low when compared to gene delivery with viral vectors. We describe recent studies with a combination of techniques (synchrotron x-ray diffraction for structure determination, laser-scanning confocal microscopy to probe the interactions of CL-DNA particles with cells, and luciferase reporter-gene expression assays to measure transfection efficiencies in mammalian cells), which collectively are beginning to unravel the relationship between the distinctly structured CL-DNA complexes and their transfection efficiency. The work described here is applicable to transfection optimization in ex vivo cell transfection, where cells are removed and returned to patients after transfection. CL-DNA complexes primarily form a multilayered sandwich structure with DNA layered between the cationic lipids (labeled Lα C). On rare occasions, an inverted hexagonal structure with DNA encapsulated in lipid tubules (labeled HII C) is observed. A major recent insight is that for Lα C complexes the membrane charge density σM of the CL-vector, rather than the charge of the cationic lipid alone, is a key universal parameter that governs the transfection efficiency of Lα C complexes in cells. The parameter σM is a measure of the average charge per unit area of the membrane, thus taking into account the amount of neutral lipids. In contrast to Lα C complexes, HII C complexes containing the lipid 1,2-dioleoyl-sn-glycerophosphatidylethanolamine (DOPE) exhibit no dependence on ?M. The current limiting factor to transfection by cationic lipid vectors appears to be the tight association of a fraction of the delivered exogenous DNA with cationic cellular molecules, which may prevent optimal transcriptional activity. Future directions are outlined, which make use of surface-functionalized CL-DNA complexes suitable for transfection in vivo.

Cationic lipid-DNA complexes, often referred to as lipoplexes, are formed spontaneously in aqueous solutions upon mixing DNA and liposomes composed of cationic and nonionic lipids. Understanding the mechanisms underlying lipoplex formation, structure and phase behavior is crucial for their further development and design as non-viral transfection vectors in gene therapy. From a physical point of view, lipoplexes are ordered, self-assembled, composite aggregates. Their preferred spatial geometry and phase behavior are governed by a delicate coupling between the electrostatic interactions which drive lipoplex formation and the elastic properties of the constituent lipid layers, both depending on the molecular nature and composition of the lipid mixture. In this review we outline some recent efforts to model the microscopic structure, energetic and phase behavior of cationic lipid-DNA mixtures, focusing on the two principal aggregation geometries: the lamellar (Lα C), or “sandwich” complexes, and the hexagonal (HII C), or “honeycomb” complexes. We relate the structural and thermodynamic properties of these two “canonical” lipoplex morphologies to their appearance in phase diagrams of DNA-lipid mixtures, emphasizing the crucial role fulfilled by the molecular packing characteristics of the cationic and neutral lipids, as reflected in the curvature elastic properties of the mixed lipid layer.

Cationic non-viral DNA vectors are very successful in in vitro transfections but less efficient in in vivo tests. This seems mainly due to the cationic nature of the molecules used to complex DNA. In this article, we describe the design and the route towards the realization of a non-viral non-cationic vector. The strategy follows three steps: first, the incorporation of DNA to a lamellar phase; second, the making of multilamellar vesicles containing a high loading of DNA by shearing the lamellar phase and, finally, the grafting of peptides onto the surface of the vesicles to target a specific receptor on the cells. Throughout this process, we had to overcome many obstacles; this review describes the present state of our work and summarizes the remaining steps.

Non-viral (synthetic) nucleic acid delivery systems have the potential to provide for the practical application of nucleic acid-based therapeutics. We have designed and prepared a tunable, non-viral nucleic acid delivery system that self-assembles with nucleic acids and centers around a new class of polymeric materials; namely, linear, water-soluble cyclodextrin-containing polymers. The relationships between polymer structure and gene delivery are illustrated, and the roles of the cyclodextrin moieties for minimizing toxicity and forming inclusion complexes in the self-assembly processes are highlighted. This vehicle is the first example of a polymer-based gene delivery system formed entirely by self-assembly.

Assembling structures to divide space controllably and spontaneously into subunits at the nanometer scale is a significant challenge, although one that biology has solved in two distinct ways: prokaryotes and eukaryotes. Prokaryotes have a single compartment delimited by one or more lipid-protein membranes. Eukaryotes have nested-membrane structures that provide internal compartments - such as the cell nucleus and cell organelles in which specialized functions are carried out. We have developed a simple method of creating nested bilayer compartments in vitro via the “interdigitated” bilayer phase formed by adding ethanol to a variety of saturated phospholipids. At temperatures below the gel-liquid crystalline transition, ™, the interdigitated lipid-ethanol sheets are rigid and flat; when the temperature is raised above ™, the sheets become flexible and close on themselves and the surrounding solution to form closed compartments. During this closure, the sheets can entrap other vesicles, biological macromolecules, or colloidal particles. The result is efficient and spontaneous encapsulation without disruption of even fragile materials to form biomimetic nano-environments for possible use in drug delivery, colloidal stabilization, or as microreactors. The vesosome structure can take full advantage of the 40 years of progress in liposome development including steric stabilization, pH loading of drugs, and intrinsic biocompatibility. However, the multiple compartments of the vesosome give better protection to the interior contents in serum, leading to extended release of model compounds in comparison to unilamellar liposomes.

HIV-1 and other complex retroviruses express six auxiliary genes in addition to the canonical retroviral genes, gag, pol and env. Vif (virion infectivity factor) protein is absolutely essential for productive HIV-1 infection of peripheral blood lymphocytes and macrophages, the two major HIV-1 target cells in vivo. However, Vif is not required for production of infectious particles in several human cell lines. In spite of the prominent phenotype of Vif mutations, the mechanism of its action remains unknown. During the last decade several models were suggested to explain the mechanism of Vif activity. One view holds that Vif is active in virions after budding or after entry into target cells during the early stages of HIV-1 replications. The second view places the action of Vif at the late stage of HIV-1 replication in virus producing cells, which affects the production of infectious virus. According to this view, Vif either compensates the cell factor required for production of infectious virus, or alternatively, it neutralizes a cell factor, which prevents the production of infectious particles in these cells. This review is addressed to summarize the models envisioned to explain Vif activities. The findings described here, that Vif interacts with viral and cellular components, elaborates the importance of Vif as a novel target for developing anti HIV-1 drugs.

The assessment of mitochondrial respiratory chain enzyme activity in human samples is a difficult task due to both the small amount of tissue generally available and the frequent need to perform enzyme activity measurement in crude mitochondrial fraction. This is particularly true for the measurement of complex III activity which partial deficiency can be easily overlooked. In this review, we first consider the several interfering reactions occurring when measuring this activity. We subsequently describe the use of an alkyl glycoside detergent, lauryl maltoside, to keep these interfering reactions to a very low level. Next, we quantify the effect of the detergent on the actual measurement of complex III in various human tissue samples and cells. Finally, we also demonstrate that the use of the detergent allows (i) a better detection of an inherited partial defect affecting cytochrome b, a catalytic subunit of the mitochondrial complex III, (ii) to possibly discriminate decreased complex III activity resulting from an abnormal complex III assembly (BCS1 gene mutation) from an hampered catalytic activity originating from a cytochrome b mutation. This detailed review of the problems associated with complex III assessment and of their tentative solution highlights the difficulties still encountered in the measurements of mitochondrial respiratory chain in humans.

α-Galactosylceramide (α-GalCer), is a glycolipid which has been identified as a ligand recognized by a special group of immune T cells, known as invariant NKT cells. γ-GalCer can powerfully activate invariant NKT cells to produce immunoregulatory cytokines, including interferon-α and IL-4, and thereby exert a variety of subsequent effects on other cells in the immune system. Recent studies have revealed the mechanism of α- GalCer-induced iNKT cell-activation in immune responses to tumors and microbes, and in the suppression of autoimmune diseases. In this review, we discuss the potential immunomodulatory activity of α-GalCer and its possible future application for clinical studies in humans.

An important virulence factor of the pathogenic fungus Cryptococcus neoformans is its polysaccharide capsule. The capsular polysaccharides glucuronoxylomannan (GXM), galactoxylomannan (GalXM) and the mannoproteins (MPs) display various immunomodulatory effects on the host response, such as the inhibition of phagocytosis, suppression of T-cell mediated immunity, and induction of immunogenic tolerance. Moreover, these capsular polysaccharides are able to interfere with the migration of phagocytes despite adequate stimulation of chemokine production and their concerted action accounts for the mild inflammatory response often observed in cryptococcosis. Different mechanisms contribute to this phenomenon. First, cryptococcal polysaccharides impair leukocyte migration towards chemoattractants. A combination of the intrinsic chemoattracting properties of circulating polysaccharides and the ability to induce cross-desensitization of chemokine receptors prevents leukocytes from leaving the bloodstream and migrating towards inflammatory site. Polysaccharide-induced repressive effects on the C5a receptor expression on neutrophils may also add to this impaired chemokinesis. Second, polysaccharides interfere with leukocyte adhesion to and migration through the endothelium. Both GXM and MP-4 induce L-selectin shedding from the surface of leukocytes; hence, interference with leukocyte rolling on the endothelium can be expected. GXM also interferes with the subsequent process of firm leukocyte adhesion to the endothelium in vitro. Thirdly, capsular polysaccharides enhance the production of anti-inflammatory interleukin-10 (IL-10) and induce tumor necrosis factor-alpha (TNFα) receptor loss from the surface of neutrophils. The capacity to reduce neutrophil influx makes cryptococcal polysaccharides interesting compounds to study in clinical models of inflammation (i.e.; sepsis, auto-immune disorders) in which leukocyte influx can be potentially damaging to host tissues.