Naked DNA

Naked DNA possesses little-to-no ability to transfect mammalian cells, with the exception of muscle cells [1]. Physical methods such as direct microinjection and 

The in-vivo disposition and tissue distribution of naked plasmid DNA has been studied by several investigators after administration via various routes, including intravenous (IV), intramuscular (IM), intradermal, and intranasal. It is generally difficult to determine pharmacokinetic parameters for naked DNA as it is rapidly and extensively degraded in plasma. Our studies indicated that supercoiled DNA is degraded with a half-life of 1.2 min upon incubation in isolated rat plasma at 37 °C [3], The open-circular form has a half-life of 21 min, while the linear form degrades with a half-life of 11 min. Other studies show similar results, with most of the supercoiled form of plasmid converting to open-circular and linear forms by 15 min [4], The 

Liu et al. [7] found significant differences between the disposition of complexed and uncomplexed [1251]-labeled plasmid DNA in mice. An equal amount of radioactivity was found in blood and liver at 15 min after injection of naked plasmid DNA. In contrast, at the same time-point, a major portion of injected DNA was in the lung when DNA-lipid complexes were injected. 

---

Plasmids can be introduced into ceUs by several methods. The most common method is transformation, where the recipient ceUs are made competent to receive DNA by washing with a solution of or other inorganic ions. Then the naked DNA is added direcdy a fraction of the ceUs take... 

A third mechanism of plasmid transfer is by transformation, which is the ability of certain microorganisms to acquire naked DNA from the environment. This is limited to certain bacteria, notably Neisseria gonorrhoeae, which is naturally competent to acquire DNA in this manner. Neisseria gonorrhoeae strains have the ability to recognize DNA from their own species, and are thus selective in their acquisition of naked DNA from the environment. 

An important consideration for DNA CT in the cell is the dramatically different environment of the DNA molecule in vivo. Unlike the naked DNA typically used in our in vitro assemblies, cellular DNA is intimately associated with proteins, packaged into chromosomes, and stored in the cell nucleus. Several experimental investigations demonstrate definitively that proteins can both modulate and participate in CT reaction in DNA. 

Higher expression levels were associated with the encapsulated DNA than with the administration of naked DNA in vitro, but standard liposomal transfection yielded the highest level of gene expression. In vivo, however, the encapsulated DNA exhibited higher levels of sustained expression of a marker gene at 28 days, than that associated with naked DNA or DNA delivered with liposomes. Thus, this study indicated the potential for DNA encapsulated in degradable nanospheres to elicit sustained gene expression in vivo. 

Once assimilated by the cell, the exogenous nucleic acid must now travel/be delivered to the nucleus. In some cases, the mechanism by which this transfer occurs is understood, at least in part (e.g. in the case of retroviral vectors). In other cases (e.g. use of liposome vectors or naked DNA), this process is less well understood. At a practical level, gene therapy protocols may entail one of three different strategies (Figure 14.3). 

Adeno-associated virus Direct injection of naked DNA... 

Up until this point, it was assumed that naked DNA injected into animals would not be spontaneously taken up and expressed in host cells. This finding vindicated the cautious approach taken by the FDA and other regulatory authorities with regard to the presence of free DNA in biophar-maceutical products (Chapter 7). 

DNA-viruses with envelopes, which are solvent-sensitive are less affected by freeze drying than solvent-resistant, naked DNA viruses. 

Since the uptake of particles in nasal epithelial tissue is known to be mostly mediated by M cells, nasal administration has been investigated as a noninva-sive delivery of vaccines [37], However, since the uptake of naked DNA by endocytocis is limited, use of either nanoparticles as mucosal delivery systems [37] or hypotonic shock [38] is reported for the efficient transfection of gene and vaccine into the nasal epithelium. It was also reported that polypeptides and polypeptide-coated nanospheres (diameter about 500 nm) are transported through endocytic process in rat M cells [39],... 

Retrovirus Adenovirus Adeno- Associated Virus Liposomes Naked DNA... 

Genes can be introduced by the application of naked DNA alone however, better efficiency is achieved when the DNA is incorporated into a delivery vector. These delivery vectors consist of viral, those utilizing modified virus particles for DNA delivery, and nonviral, for which various chemicals are used to aid DNA packaging and delivery. Viral vectors confer significantly better transfection efficiency than nonviral vectors however, recently the toxicity and oncogenic side effects of viral vectors have become a major concern (6). Nonviral vectors do not have such serious side effects but lack the efficiency (7). 

BT 20 cells incubated in serum free medium for 10 hours with the vector/ DNA complexes (DQAplexes, C-DQAplexes). For control, cells were exposed to naked DNA and empty vesicles. The cells were then stained with Mitotracker Red CMXRos (Molecular Probes) for five minutes to enable the visualization of mitochondria followed by confocal fluorescence microscopic analysis on a Zeiss Meta 510 Laser Scanning Microscope. 

A prerequisite to these calculations is the knowledge of bending and torsional rigidity coefficients (A and C, respectively) of the naked DNA. A was calculated from a persistence length a = A/kT = 50 nm [58], and C through the following equation, valid only for a naked DNA minicircle near relaxation [59]... 

---