Plasmid-based gene delivery

Nonviral vector systems are usually either composed of a plasmid based expression cassette alone ( naked DNA), or are prepared with a synthetic amphipathic DNA-complexing agent (84, 88). Gene delivery systems based on nonviral vectors mainly comprise cationic liposomes, DNA-polymer-protein complexes, and mechanic administration of naked DNA. An idealized/optimized multifunctional nonviral gene delivery system is depicted in Figure 13.4. 

The elements of a nonviral gene delivery system include a gene coding for the therapeutic gene, a plasmid-based expression system, and a synthetic delivery system. 

Singh R, Pantarotto D, McCarthy D, Chaloin O, Hoebeke J, Partidos CD, Briand JP, Prato M, Bianco A, Kostarelos K (2005) Binding and condensation of plasmid DNA onto functionalized carbon nanotubes Toward the construction of nanotube-based gene delivery vectors. J. Am. Chem. Soc. 127 4388 4396. 

The major problem associated with plasmid based gene delivery to skeletal muscle is the relatively low efficiency of transfection. Recent developments have demonstrated improved delivery associated with the application of an electrical field to the muscle after injection of the plasmid DNA (109). It is clear that the application of naked DNA close to the site of pathology and away from degradative elements such as plasma is thus a viable strategy for gene delivery. However, this method is ineffective if DNA dosing to anatomically inaccessible sites (e.g., solid tumors in organs) is desired. 

Hagstrom, J.E. (2003). Plasmid-based gene delivery to target tissues in vivo the intravascular approach. Curr. Opin. Mol. Ther., 5(4), 338-344. 

The assembly of NLS in peptide-based gene delivery systems has been achieved by the non-covalent binding of plasmid to either free NLS embedded with polyplexes or to NLS linked to a cationic sequence, such as (PKKKRKV)4-K2o (Table 16.7), AKRARLSTSFNPVYPYEDES-K20 (Table 16.7) or H9-2 sequence (nls-H9-2) (Table 16.4). With nls-H9-2, the transfection efficiency with a formulation containing... 

Anti-deoxyribonucleic acid autoantibodies from human and mice suffering from Lupus erythematosus can penetrate into cells and accumulate in the cell nucleus. Based on the characteristics of a mi-ON A autoantibodies, VAYISRGGVSTYYSDTVKGRFTRQKYNKRA peptide (P3), which exhibits a-helix, has been used as a vector for the intracytoplasmic and intranuclear translocation of macromolecules (Table 16.7) (Avrameas et al., 1998, 1999). P3 shares similar capabilities with Antenapedia peptide (Derossi et al., 1994), but in contrast P3 operates only at 37 °C by an energy dependent mechanism. P3 linked to a 19 lysine residue sequence (K19-P3) forms complexes with plasmid DNA. Efficient transfection of mouse 3T3 cells and hamster lung CCL39 cells were obtained with these complexes. This transfection was not impaired by the presence of serum and did not require helper molecules such as chloroquine. These observations suggest that peptides from cell specific anti-DNA autoantibodies may represent a source of peptide-based gene delivery system with different specificities. 

A current theme in plasmid-based delivery approaches is to mimic Nature s methods for nucleic acid delivery. To date, the best system to emulate Nature has been viral vectors. Briefly, most viral vectors escape immune surveillance, interact with cell membranes (e. g., receptor), internalize (via endocytosis), escape from endosomes, migrate to the nuclear envelope, enter the nucleus, and finally take over cellular functions. Plasmid-based systems (cationic liposomes and cationic polymers) can mimic portions of these events. This chapter will explore the barriers facing gene delivery vectors, with an emphasis of the pharmacokinetic behavior of these systems. In order to understand the in-vivo barrier, a brief review of physiology will be provided. 

Figure 14.4 Fate of plasmid DNA after in vivo administration (modified with permission from Site-specific gene therapy design and use of plasmid-based gene medicines to control in vivo production, delivery and effect of therapeutic proteins, Eric Tomlinson (1998). In Peptide and Protein Drug Delivery, Alfred Benzon Symposium 43."...

Describe the pharmacodynamic and pharmacokinetic barriers to effective plasmid-based gene delivery. 

Describe the use of plasmid-based gene delivery for vaccination. 

Transdermal vaccination or transcutaneous immunization, is attractive, because it does not require specially trained personnel necessary for needle injections. Topical application of antigens to intact skin has shown promising results for the administration of DNA-based vaccines. Noninvasive gene delivery by pipetting adenovirus- or liposome-complexed plasmid DNA onto the outer layer of skin was able to achieve localized transgene expression within a restricted subset of skin in mice. It also elicited an immune response against the protein encoded by the DNA. ... 

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...

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