Current Medicinal Chemistry - Volume 10, Issue 14, 2003

Completion of the working draft of human genome has unquestionably strengthened the clinical potential of gene therapy. The principle of gene therapy is simple: “Supplement the body cells with the corrected copies of the malfunctioning genes”. However, given that both the macromolecular genes (DNA) and biological cell surfaces are negatively charged, spontaneous entry of naked DNA inside cells are unlikely to be an efficient process. Thus, efficient delivery and expression of genes into body cells (a process biologists call “transfection”) is always easier said than done. In other words, the problems of developing clinically viable gene therapeutic approach and designing safe and efficient gene delivery reagents are inseparable: shortcomings in one is going to adversely affect the success of the other. Clearly, the realization of the full potential of gene therapy will critically depend on the future development of safe and efficient gene delivery reagents. Transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral vectors are, in general, superior to their nonviral counterparts. However, potential adverse immunogenic aftermath associated with the use of viral vectors such as on-set of serious immune response to the expressed viral proteins, innate humoral response, possibility of random integration into the host chromosome & subsequent activation of proto-oncogenes, possibility of systemic clearance due to complement activation, etc. are increasingly making non-viral cationic transfection lipids as the vectors of choice. The cationic transfection lipids have come a long way in its voyage to clinical success. Initial excitements in late eighties and early nineties were followed by a period of set-backs during late nineties in developing clinically viable & systemically stable lipoplexes (complex of lipids and DNA). Breakthrough technical advances in designing stable lipoplexes capable of successfully confronting the daunting systemic challenges are increasingly being reported with the dawning of the new millennium. The guest editor firmly believes that the articles of the present theme issue written by the experts in the field will provide genuine impetus to more and more creative minds across the scientific disciplines towards ensuring clinical success of cationic transfection lipids in gene therapy. At the end, my heart-felt thanks to all the invited contributors for spending so much of their time and efforts for this theme issue.

Successful gene therapy depends on efficient gene transfer vectors. Viral vectors and non-viral vectors have been investigated extensively. Cationic lipids are non-viral vectors, which resemble traditional pharmaceuticals, display little immunogenicity, and have no potential for viral infection. However, toxicity and low transfection efficiency are two barriers limiting the clinical applications of cationic lipids. Over the last decade, hundreds of cationic lipids have been synthesized to address these problems. In this brief review, we summarized recent research results concerning the structures of DNA / liposomes complexes, some important strategies used to design different classes of cationic lipids, and use of disulfide cationic lipids in plasmid DNA delivery.

Gene therapy research is in crisis owing to the lack of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but currently appear to be too dangerous for routine clinical use. Synthetic non-viral vectors are inherently much safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors based upon well-found platform technologies and a thorough understanding of the barriers to efficient gene delivery and expression (transfection) relevant to clinical applications of interest. In this review, the current status and prospects for cationic liposome / micellebased synthetic non-viral vector systems are discussed including a description of the barriers to efficient transfection, a summary of the main structure / activity studies and mention of ternary cationic liposome / micelle-nucleic acid (LD) systems. The review culminates with a description of two promising cationic liposome / micelle-based non-viral vector platform systems known as liposome:mu:DNA (LMD) and stabilised plasmid-lipid particles (SPLP) that should create a real opportunity for the development of clinically viable synthetic vector systems within the next few years.

Cationic liposome-DNA complexes, also called “lipoplexes”, constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. Here we review the mechanisms of lipoplex-mediated gene delivery, the barriers to efficient gene expression, and novel cationic lipids used for transfection. We also describe methods for enhancing gene transfer via the use of proteins, including transferrin, albumin and asialofetuin, and synthetic peptides, including GALA and nuclear localization signal peptides. We underscore the importance of understanding the mechanisms of cytoplasmic and nuclear entry of DNA and its dissociation from lipoplexes. We emphasize that the in vitro transfection activity of new lipoplex constructs should be tested in the presence of high serum concentrations to emulate in vivo conditions.

The use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of gene therapy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. Despite extensive research in the last decade on the use of cationic liposomes as gene transfer vectors and the development of elegant strategies to enhance their biological activity, these systems are still far from being viable alternatives to the use of viral vectors in gene therapy. In this review considerations are made regarding the structure-activity relationships of cationic liposome / DNA complexes and the key formulation parameters influencing the features of lipoplexes are presented and discussed in terms of their effect on biological activity. Particular emphasis is given to the interaction of the lipoplexes with serum components as well as to novel strategies developed to circumvent difficulties that may emerge upon iv administration of the complexes. Finally, since the ability of the lipoplexes to be stored while preserving their transfection activity is a crucial issue for the repeated use of such carriers, approaches reported on the improvement of their physical stability are also reviewed.

Non-viral synthetic vectors for gene delivery represent a safer alternative to viral vectors. Their main drawback is the low transfection efficiency, especially in vivo. Among the non-viral vectors currently in use, the cationic liposomes composed of cationic lipids are the most common. This review discusses the physicochemical properties of cationic lipids, the formation, macrostructure and specific parameters of the corresponding formulated liposomes, and the effect of all these parameters on transfection efficiency. The optimisation of liposomal vectors requires both the understanding of the biological variables involved in the transfection process, and the effect of the structural elements of the cationic lipids on these biological variables. The biological barriers relevant for in vitro and in vivo transfection are identified, and solutions to overcome them based on rational design of the cationic lipids are discussed. The review focuses on the relationship between the structure of the cationic lipid and the transfection activity. The structure is analysed in a modular manner. The hydrophobic domain, the cationic head group, the backbone that acts as a scaffold for the other domains, the linkers between backbone, hydrophobic domain and cationic head group, the polyethyleneglycol chains and the targeting moiety are identified as distinct elements of the cationic lipids used in gene therapy. The main chemical functionalities used to built these domains, as well as overall molecular features such as architecture and geometry, are presented. Studies of structure-activity relationships of each cationic lipid domain, including the authors', and the trends identified by these studies, help furthering the understanding of the mechanism governing the formation and behaviour of cationic liposomes in gene delivery, and therefore the rational design of new improved cationic lipids vectors capable of achieving clinical significance.

Among other strategies, the use of cationic lipids as autoassembling vehicles for non viral DNA transfection has received considerable attention. An exponentially growing litterature has been published on this topic (over 700 hits for the past decade, including 400 in the last two years). The present review focuses on the main present strategies aiming at improving cationic lipids induced transfection, and on some of the frequently encountered problems that should be solved to apply these non-viral vectors for human health. The review contains several sections dealing with the chemistry, physico-chemistry, cell biology, in vivo biology, and targeting of cationic-lipid DNA complexes.

Optimization of cationic liposomal complexes for in vivo applications is complex involving many diverse components. These components include nucleic acid purification, plasmid design, formulation of the delivery vehicle, administration route and schedule, dosing, detection of gene expression, and others. This review will primarily focus on optimization of the delivery vehicle formulation. These formulation issues include morphology of the complexes, lipids used, flexibility versus rigidity, colloidal suspension, overall charge, serum stability, half-life in circulation, biodistribution, delivery to and dissemination throughout target tissues. Optimizing all components of the delivery system will allow broad use of liposomal complexes to treat or cure human diseases or disorders.

The present article reviews interesting cationic liposomes (cationic transfection lipids) with novel cationic cholesterol derivatives, a new strategy in gene transfection developed by our group and the presently accepted molecular mechanism of gene transfection. Use of confocal laser scanning microscopy and atomic force microscopy in elucidating the molecular mechanism of gene transfection by cationic liposomes is also reviewed using examples from our own work. As delineated below, both the confocal laser scanning microscopic and the atomic force microscopic results advocate for the involvement of the sequential three steps in gene transfection mediated by the cationic liposomes: endocytotic internalization of the lipoplexes (liposome-DNA complexes) into the target cells, endosome-lysosome fusion whereby the DNA gets released from the liposomes and moves towards the nucleus of the target cells and microtubule organization apparently involved in trafficking the transfected foreign genes to lysosomes. Furthermore, the present article also reviews couple of important strategies in gene transfection namely, use of liposomes made from biosurfactants and harnessing efficient gene transfection by activating the membrane-bound receptor molecules.

The clinical success of gene therapy is critically dependent on the development of efficient and safe gene delivery reagents, popularly known as “Transfection Vectors”. The transfection vectors commonly used in gene therapy are mainly of two types: viral and non-viral. The efficiencies of viral transfection vectors are, in general, superior to their non-viral counterparts. However, the myriads of potentially adverse immunogenic aftermaths associated with the use of viral vectors are increasingly making the non-viral gene delivery reagents as the vectors of choice. Among the existing arsenal of non-viral gene delivery reagents, the distinct advantanges associated with the use of cationic transfection lipids include their: (a) robust manufacture; (b) ease in handling & preparation techniques; (c) ability to inject large lipid:DNA complexes and (d) low immunogenic response. The present review will highlight the successes, set-backs, challenges and future promises of cationic transfection lipids in non-viral gene therapy.

This review focuses on the recent developments in study of cationic lipids as carriers for DNA delivery. Emphasis is placed on a class of compounds as exemplifies by their similarity in structures and transfection activities. The technical aspects are also reviewed on how to prepare DNA-lipid complexes and to perform transfection. A brief discussion of the current views on the mechanism of cationic lipid-mediated DNA transfer is intended to provide new prospects for future developments and further improvement of the current systems.