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Non-viral DNA delivery systems
It is known that about 75% of recent clinical protocols- involving gene therapy make use of viral-based vectors for DNA delivery. However, in clinical trials, none of these viral vectors could be shown to be safe. Therefore, non-viral DNA delivery systems have been developed both for gene therapy and DNA vaccination for treating and controlling diseases.

Most of these available DNA delivery systems are though versatile and safe, they are less efficient than the virus-mediated DNA delivery systems discussed above. The low efficiency of non-viral DNA delivery is due to the following three major barriers to DNA delivery: (i) low uptake across plasma membrane, (ii) inadequate release of DNA molecules and (iii) lack of nuclear targeting. These three barriers have been addressed in the efforts to develop efficient DNA delivery systems, which can be broadly classified into (i) mechanical/electrical methods and (ii) chemical methods.

Mechanical/electrical methods. Direct injection of naked DNA into the cell is simple and therefore appealing, although there are problems associated with it, since only single cells can be manipulated, and the process is slow and laborious. The three methods that have been tried include: (i) low voltage electroporation or microinjection; (ii) particle bombardment and (iii) pressure-mediated DNA delivery. The details of electroporation and particle bombardment will be discussed in Chapter 33, and are commonly used with cultured cells  except where limited local expression of delivered DNA is adequate to achieve immune response (e.g., DNA vaccination in epidermal or muscle cells). Pressure-mediated delivery gave 50% efficiency of delivery, when tried with cardio-vascular tissue. Hydrodynamic force has also been used for DNA delivery to hepatocytes with 40% efficiency. Ultrasonic nebulization has also been tried.

Chemical methods. The use of a chemical to enhance DNA uptake by the cell is considered easy and versatile. The techniques can be broadly classified into those based on : (i) 2 diethylamino ether (DEAE)-dextran, (ii) calcium phosphate, (iii) artificial lipids, (iv) proteins, (v) dendrimers, etc. In all these techniques, DNA is complexed with a chemical and is deposited onto the cells, so that they are internalized by endocytosis and the DNA is released at the desired target site. The major limitation of these methods is toxicity upon systemic administration.

(a) Efficient uptake of DNA during a pulse of DNA complexed with a chemical. During 1965-75, DEAE-dextran-DNA and calcium phosphate-DNA complexes were used for internalization by the cells through endocytosis. These methods can be largely used for in vitro transfection and suffer with problems of unstable and variable transfection. During 1970-1990, a variety of artificial lipids-based DNA delivery systems were developed, which are the most commonly used methods today. Lipofectin-DNA complexes were the first chemical systems that could be used in animals and liposome-mediated DNA delivery has been used in humans. The cationic peptide poly-L lysine (PLL), polyamidoamine (PAMAM) dendrimers and polyethyleneimine (PEl) are other chemicals, which vary in efficiency (20%-80%), but are toxic.

(b) DNA complexed with polymers for controlled release on a Iong -term basis. The above chemical methods involve exposure of cells to a pulse of DNA complexed with a chemical, so that a short period is available for uptake of DNA. Therefore, biocompatible polymers have been used, which allow controlled release of DNA on a long-term basis without repeat administration. For instance, biodegradable poly (D) L-lactide-co-glycolide (PLG), microparticles/microspheres and poly ethylene-co-vinyl acetate (EV Ac) matrices have been used for long-term controlled release of naked DNA within the cell.

 
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