Missing pieces in understanding the intracellular trafficking of polycation/DNA complexes.

In about 70% of over 1400 gene therapy clinical trials that have been conducted to date worldwide, genetically-modified viruses have been the carrier of choice for delivery of therapeutic genetic material [1]. While the viruses promise both high efficiency of transfer and great protection of the therapeutic genes [2], this approach also carries a risk of causing adverse (inflammatory or immune) reactions [3,4] or even cancer [5]. Non-viral systems, such as cationic lipids and synthetic polymers (in particular, polycations), have attracted the interest of a large number of researchers as safer alternatives [6]. In particular, polycations have become popular components of non-viral gene carriers because of the relative ease with which their chemical and physical properties can be engineered for specific applications. However, the polycation-based approach has been limited in its clinical application in large part due to the poor biological activities of synthetic polymers on both cellular and systemic levels. A major issue is the difficulty associated with target-cell-specific delivery of genetic materials in vivo [6,7]. However, even the basic problem of achieving a sufficient efficiency in the transportation of therapeutic genes across various intracellular barriers also remains one of the leading challenges in the development of superior polycation-based gene delivery systems. In this regard, even the most effective polycation gene carrier (e.g., linear polyethylenimine or PEI for short) remains 10 times less efficient [8] than its viral counterpart [9]. Since the first demonstration of polycation-medicated gene transfection in 1987 [10], many polycation materials (both new and off-the-shelf) have been explored for gene delivery applicationswith themost intensively studied example being the PEI polycation (reviewed in Refs. [11–16]). An obvious reason for the great attention devoted to PEI is that this polycation affords the highest levels of in vitro gene transfection. It is believed that the high gene transfection efficiency observed with PEI is attributable to its unique ability to simultaneously overcome several key barriers to intracellular trafficking of the DNA particles (e.g., escape from endosomes [17,18], protection of DNA from degradation by endonulceases [19], nuclear entry [17,19,20], DNA release and transcription [20]). Currently, however, the exact mechanisms of how PEI orchestrates the sequence of the intracellular processes required for effective expression of the transgene in the host cell, and the particular chemical/molecular attributes of PEI responsible for each event, remain largely unexplained, making it difficult to further improve the performances of the PEI-based carriers in other aspects of the delivery process. One recent example to improve the PEI-based delivery system is the incorporation of intracellularly degradable disulfide bonds in the backbone structure of the PEI molecule [21–24] to reduce the inherent cellular (and systemic) toxicity of the polycation [25–27]. While this modification improves the viability of the transfected cells, thereby enabling the use of the PEI chemistry at high molecular weight without causing cell death [22–24], this improvement accompanies an unwanted decrease in the overall gene transfection efficiency when the performances are compared at an identical PEI molecular weight [24]. Improved understanding of the polycation chemistry vs. performance mechanism relationships will provide useful insights to guide further (chemical and/or physical) modifications of this already useful polycation toward creating multipotent gene carriers that can accommodate all of the sophisticated functional requirements at various stages of the delivery process. In this article, we intend to identify and discuss several key areas which require further improvements in our molecular understanding of the cellular transport processes of polymer/DNA complexes (“polyplexes”).