Gene transfection, mechanism

Three families of polymers have been used to study transfection mechanisms polyamines, polyamides, and polyvinyl type polymers. The transfection efficiencies achievable with these systems vary widely, so an in-depth analysis of each polymer family and subsequent comparison of what affects gene delivery will be discussed in this chapter. In addition to high transfection efficiency, it is important for the polymeric systems to be relatively nontoxic to cells in vitro and not to elicit an immune response in vivo. Thus, the effect of transfection parameters on cytotoxicity and immunogenicity will also be examined. 

Fig. 1 Proposed mechanism of gene transfection [117]. (1) Formation of the DNA/polymer complex (polyplex), (2) endocytosis of the polyplex, (3) fusion of endosome and lysosome, (4) release of the polyplex into the cytosol, (5a) incorporation of the polyplex into the nucleus, (5b) release of the siRNA into the cytosol, (6) transcription of the DNA into mRNA followed by release of the polyamine back into the cytosol, (7a) translation of mRNA, and (7b) mRNA degradation. The metabolism of the polyamine is still unclear. Reproduced with permission from [117]. Copyright 2002 Elsevier...

Therefore, improved chemical synthesis strategies for the design of tailored polymeric gene vectors and more information on transfection mechanisms will be needed for the optimization of DNA and siRNA vehicles. 

Fig. 3. Property of gene delivery with BLs and US exposure (a) Schema of transfection mechanism by BLs and US. The mechanical effect based on the disruption of BLs by US exposure, which results in generation of some pores on plasma membrane, is associated with direct delivery of extracellular plasmid DNA into cytosol, (b) Luciferase expression in COS-7 cells transfected by BLs and US. COS-7 cells (1x10 cells/500 pLAube) were mixed wifh pCMV-Luc (5 pg) and BLs (60 pg). The cell mixture was exposed with US (Frequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/ cm. Time 10 s). The cells were washed and cultured for 2 days. Affer fhaf, luciferase acfivify was measured, (c) Effecf of US condition on transfection efficiency with BLs. COS-7 cells were exposed with US (Frequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/cm Time 0,1, 5,10 s) in the presence of pCMV-Luc (0.25 pg) and BLs (60 pg). Luciferase activity was measured as above, (d) Effect of serum on transfection efficiency of BLs. COS-7 cells in the medium containing EBS (0,10, 30, 50% (v/v)) were treated with US (Erequency 2 MHz, Duty 50%, Burst rate 2 Hz, Intensity 2.5 W/cm, Time 10 s), pCMV-Luc (0.25 pg) and BLs (60 pg) or transfected with lipoplex of pCMV-Luc (0.25 pg) and lipofectin (1.25 pg). (e) In vitro gene delivery to various types of cell using BLs and US. The method of gene delivery was same as above. S-180 mouse sarcoma cells, Colon26 mouse colon adenocarcinoma cells, B16BL6 mouse melanoma cells, Jurkat human T cell line, HUVEC human umbilical endothelial cells. Luciferase activity was measured as above. <10 RLU/mg protein, <10 RLU/mg protein Each data represents the mean S.D. n=3). L PEG-liposomes, LF Lipotectin...

To develop an efficient gene delivery system, it seems necessary to understand the extra- and intracellular processes involved in the overall transfection mechanism. This will lead to understanding the mechanism, which is necessary for developing novel lipid-based non-viral vectors. For this purpose, cationic liposomes and pDNA are used widely to understand the cellular mechanism involved in the transfection (Fig. 13.3). 

Regardless of the strategy, it is imperative to understand the mechanism(s) involved in the delivery process. Targeting of DNA to a specific cell population by any method can only be optimized when the components of the delivery system are fully characterized. This will ultimately enable researchers to effectively assess the efficiency of DNA delivery and cellular uptake, critical parameters in gene transfection. It is important to determine how cells handle exogenously administered DNA and to determine where the rate-limiting step is in the transfection process. This chapter will address many of the current issues related to the development and analysis of nonviral-mediated DNA delivery methods, with emphasis on the lipid-based carrier systems. 

Finally, a range of steroidal polyamines were found to promote transport across cell/liposome membranes or bind to nucleic acids and promote gene transfection. Although these compounds have not in general been studied as receptors, their interactions with anionic centers are presumably central to their activities. For example, 22 and 23 were incorporated in vesicle membranes and shown to discharge pH gradients across the membranes. Their mechanisms of action are... 

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