Biomedical Engineering Reference
In-Depth Information
11.2.4 Non-viral DNA vectors
Research into alternative non-viral gene delivery methods has been gathering pace in recent
years, following mounting concerns into the use of viral vectors. Despite their high effi-
ciency, possible insertional activation of oncogenes or adverse immune responses pose very
real threats to the success of viral gene therapy treatments. There have been several incidents
of serious illness and death following treatment with viral vectors. These include the death
of a teenager from adverse immunological reactions after adenoviral treatment for a liver
enzyme deficiency [54] and reports of 4 out of 11 boys developing leukemia after being
treated in France with retroviruses for SCID [55]. The use of non-viral vectors offers many
safety advantages over viruses (see Table 11.1) but, to date, remains less efficient.
Non-viral vector delivery methods fall broadly into two categories: physical and chemical
methods.
1
Physical methods
: Simple direct injection of plasmid DNA, without the inclusion of
any vector, has shown remarkable success in a number of tissues, including muscle,
liver, skin and solid tumors (reviewed in Herweijer and Wolff [56]). Direct injection
into skeletal muscle shows the most promise for DNA-based immunization procedures,
although gene therapy applications are also under development.
Untargeted intravascular delivery of plasmid DNA into whole animals, either systemic
or regional, does result in low-level widespread transient transgene expression [57].
However, increasing hydrodynamic pressure by rapid delivery of large volumes of DNA
substantially increases efficiency [58], localizing largely to the liver. Transgene expres-
sion can be further improved by localized hydrodynamic delivery to specific organs,
including the liver [59] and the kidney [60].
The addition of physical methods to temporarily disrupt membranes and enhance DNA
transfer have been explored, including particle bombardment by gene gun [61], ultra-
sound [62] and electroporation [63]. Whilst all of these methods have shown successful
gene delivery, their harsh and almost always unphysiological nature lead to unwanted
cytotoxicity. Therefore, clinical application is realistically highly limited.
2
Chemical methods
: Chemical methods almost exclusively contain a polycationic com-
ponent, enabling DNA binding and condensation, as well as electrostatic interaction with
cell surface molecules.
11.2.4.1 Cationic liposomes
Liposomal gene delivery, first pioneered by Felgner and colleagues in 1987 [64], uses
cationic lipids that readily bind the negative phosphate backbone of DNA, spontaneously
condensing the DNA into small particles and protecting it from intracellular degradation
[65]. Internalization of the DNA is thought to occur via both coated pit and non-coated
endocytosis pathways, depending on the charge and size of the liposomal complexes.
A cationic lipid is fundamentally composed of four functional domains: a positively
charged head group (usually a single or multiple amine-derived group), a spacer of varying
lengths, a linker bond and a hydrophobic anchor (see Figure 11.3). The relationship between
structure and efficiency is an area of intense research [66]. Although some cationic lipids
are effective at DNA delivery alone, they are often formulated with a non-charged 'helper'
phospholipid or cholesterol to improve stability and transfection efficiency [67]. It is thought
