Non-viral gene delivery systems

Non-viral gene delivery systems 9.2.1. DNA/DNA liposome complexes 

The limitations of viral vectors in particular their relatively small capacity for therapeutic DNA, safety concerns and difficulty in targeting to specific cell types have led to the evaluation and development of alternative vectors based on synthetic, non-viral systems. Examples of some non-viral approaches to gene delivery are discussed below. 

The simplest non-viral gene delivery system simply uses naked expression vector DNA. Direct injection of free DNA into certain tissues, particularly muscle, has been shown to produce smprisingly high levels of gene expression and the simplicity ofthis approach has led to its adoption in a munber of clinical protocols. In particular, this approach has been applied to the gene therapy of cancer where the DNA can be injected either directly into the tumour or can be injected into muscle cells in order to express tumour antigens that might function as a cancer vaccine 

Although direct injection of plasmid DNA has been shown to lead to high levels of gene expression in some tissues, the level of expression in many other tissues is much lower than with either viral or liposomal vectors. Naked DNA is also unsuitable for systemic administration due to the presence of serum nucleases. As a result direct injection of plasmid DNA seems destined to be limited to only a few applications involving tissues that are easily accessible to direct injection such as skin and muscle cells. 

Cationic liposomes are also able to interact with negatively charged cell membranes more readily than classical liposomes. Fusion between cationic vesicles and cell surfaces might result in delivery of the DNA directly across the plasma membrane. This process bypasses the endosomal-lysosomal route which leads to degradation of anionic liposome formulations. Cationic liposomes can be formed from a variety of cationic lipids, and they usually incorporate a neutral lipid such as DOPE (dioleoylphosphatidyl-ethanolamine) into the formulation in order to faciUtate membrane fiision . A variety of cationic lipids have been developed to interact with 

but perhaps the best known are DOTAP (N-l(-(2,3-dioleoyloxy)-propyl)-N,N,N-tiimethylainmo-niumethyl sulphate) and DOTMA (N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride). These are commercially available Upids that are sold as in vitro transfecting agents, with the latter sold as Lipofectin. 

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Davis, M.E., Non-viral gene delivery systems. Curr. Opin. Biotechnol. 2002, 13, 128-131. 

Figure 14.10 Overview of cellular entry of (non-viral) gene delivery systems, with subsequent plasmid relocation to the nucleus. The delivery systems (e.g. lipoplexes and polyplexes) initially enter the cell via endocytosis (the invagination of a small section of plasma membrane to form small membrane-bound vesicles termed endosomes). Endosomes subsequently fuse with golgi-derived vesicles, forming lysosomes. Golgi-derived hydrolytic lysosomal enzymes then degrade the lysosomal contents. A proportion of the plasmid DNA must escape lysosomal destruction via entry into the cytoplasm. Some plasmids subsequently enter the nucleus. Refer to text for further details...

CNTs with different characteristics, which will lead to differences in the mechanism of CNT metabolism, degradation or dissolution, clearance and bioaccumulation. On the other hand, most non-viral gene delivery systems today suffer from both limited levels of gene expression and an unfavourable toxicity profile due to their highly cationic surface character. Therefore, opportunities for CNT-based gene transfer systems are still ample. 

Kwok, K.Y., Yang, Y.S. and Rice, K.G. (2001) Evolution of cross-linked non-viral gene delivery systems. Curr. Opirt. Mol. Ther., 3,142-146. 

The addition of NLS in a non viral gene delivery system is expected to favor the nuclear import (nuclear pores allow the passage of particles of 24 nm) by using the karyopherin and importin machinery. Where should the NLS be linked, on the pDNA or on the carrier For the latter strategy, polyplexes must not dissociate before their import in the cell nucleus. Both strategies have been prospected but with only limited success. 

Tomlinson, E. and Rolland, A.P. (1996) Controlled gene therapy pharmaceutics of non-viral gene delivery systems. J. Control. Release, 39, 357-372. 

Figure 18.1 Types of non-viral gene delivery systems.

An alternative non-viral gene delivery system has been developed that uses polymers, either... 

For efficient gene delivery into the nucleus of target cells, the non-viral vectors must overcome several barriers, such as the plasma membrane, the endosomal membrane, and the nuclear membrane. Thus, to overcome the barriers, the non-viral gene delivery system must be equipped with various functional devices such as hgands for specific receptors, pH-sensitive fusogenic peptides for endosomal escape, and a nuclear locahzation signal (NLS) for enhanced nuclear dehvery [1]. 

We recently proposed a novel non-viral gene delivery system multifunctional envelope-type nano device (MEND) to realize Programmed Packaging [1-3]. The ideal MEND (see Fig. 8.1) consists of a condensed DNA core and a lipid envelope structure equipped with the various func-... 

Fewell J G, et al. (2005). Synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer. J. Control. Rel. 109 288-298. 

Lungwitz U, Breunig M, BlunkT, et al. (2005). Polyethylenimine-based non-viral gene delivery systems. Euro. J. Pharm. Biopharm. 60 247-266. 

Rolland A, Feigner PL (1998) Non-viral gene delivery systems - preface. Adv Dmg Deliv Rev 30 1-3... 

Cationic Polymer-Based Non-viral Gene Delivery Systems and their Application in Gene-Engineered Stem Cells... 

Cationic Polymer-Based Non-viral Gene Delivery Systems... 

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