Gene Therapy for the Enhancement of Fracture Healing - Engineered Bone: Tissue Engineering Intelligence Unit - page 134

Naked DNA (in the form of a plasmid) can be directly injected into certain tissues, particu-
larly muscle. Direct muscle injection was shown 76 to be feasible and produces surprisingly high
levels of gene expression. In addition to the muscle, direct injection was successful in other
tissues such as liver, 77 pancreas, 78 thyroid, 79 myocard, 80 brain, 81 urological organs 82 and blood
vessel development. 83 Enhancement of the transgene expression levels were noted following
either the use of a number of muscle-specific promoters and a myosin light chain 84 or the
modifications to the polyadenylation and transcriptional termination. 85
The simplicity of this approach has led to its adoption in a number of clinical protocols. In
particular, this approach has been applied to the gene therapy of cancer where the DNA can be
injected either directly into the tumor and the DNA expression is high enough to elicit a
therapeutic response 86,87 or into muscle cells in order to express tumor antigens that might
function as a cancer vaccine. Gene expression of direct DNA injection can persist for several
months after intramuscular injection, 88,89 and does not usually undergo chromosomal integra-
tion.
Direct injection of naked DNA into the blood stream did not result in gene expression in
major organs. 90 This was due to the presence of nucleases responsible for DNA degradation
and a clearance system performed by mononuclear phagocyte with a half-life of less than 5
minutes.
Several studies have reported uptake of naked DNA in several organs but mainly the liver
when they either rapidly injected large volumes of fluids/naked DNA into the vasculature
system in order to increase the osmotic and hydrostatic pressure or occluded blood vessels to
again achieve increased pressure. 91-94 This approach of DNA introduction appears to benefit
from the use of cationic liposomes to protect the DNA from degradation and also from in-
creased osmotic and hydrostatic pressure. A better understanding of the mechanism of uptake
of DNA by these approaches might yield more enthusiasm for its use clinically.
The Gene Gun approach uses gold particles (1-3 µ m) that are attached to DNA that are
cannonaded into the tissue. 95,96 The prolonged and sustained low level expression of this tech-
nique has made it one of the methods of choice for the development of DNA vaccines. DNA
vaccines promise to be valuable where responses to agents are inappropriate, ineffective or even
lacking, for example, HIV and influenza. 97,98 Some of the advantages of the DNA vaccines
compared to conventional attenuated and protein based vaccines are the relatively low cost to
produce, and the ability to introduce multiple pathogens by using a single plasmid. Moreover,
DNA vaccines are unaffected by preexisting immunity. 99 An interesting aspect of this method
is the likely means by which DNA vaccines induce cytotoxic T lymphocytes (CTL) and that
the type of immunity induced can be modified. This might be a practical approach to manipu-
late undesired immune response. 99
Electroporation has been widely used to transfect DNA to cells in tissue culture. 100 This
technique relies on the fact that electric pulses can induce opening up the pores in the cell
membrane through which DNA can pass down a concentration gradient into the interior of
the cell.
In vivo, naked plasmid DNA is usually injected into the interstitial space of the tissue and
then electric pulses are applied with needle or caliper-type electrodes to transfect the DNA
intracellularly. So far, this technique has been applied to introduce plasmid DNA into the
following tissues: skin, 101 liver, 102 melanoma, 103 and muscle. 104
Cationic liposomes are lipid bilayers entrapping a fraction of aqueous fluid. Cationic lipids
are widely used in vitro and the majority of cell lines may be transfected. The negatively charged
nucleic acids interact with the positively charged macromolecules, induce a condensation of
the DNA and a physical association with the lipid. Several kinds of cationic lipid, such as
dioctadecylamido-glycylspermine (DOGS, Transfectam™) 105 and Lipofectin™, 106-108 have
been developed. In addition a neutral lipid such as dioleoylphosphatidylethanolamine (DOPE)
or cholesterol is generally added to facilitate the release of plasmid DNA from the endosome
following endocytic uptake of the complex. 109 The exact mechanism of the cellular membrane
interaction with the complex and subsequent cell entry is unknown. But it is thought that