Gene Delivery Using Physical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 84

[2] . This leads to significant savings in the cost of treatment and will also reduce any
potential risk associated with the use of large amounts of DNA. Most of the physical
methods involve the use of some type of instrument, which leads to more quantita-
tive and standardized gene delivery. Such an instrumental approach is important for
large-scale clinical applications and will minimize the “common operator variation”
frequently observed with nonphysical methods in the stage of laboratory research.
This chapter describes the delivery of DNA by various physical methods, such as
electroporation (EP), sonoporation, microinjection, particle bombardment (gene gun),
and hydrodynamic injection with application of external physical force (pressure,
sound, shock wave, electric pulses, etc.) for efficient gene transfer inside organs and
cells. It also discusses methods for achieving higher gene transfection at the desired
site than those achieved with nonphysical methods of administering a similar dose of
DNA. Further, this chapter provides an overview of the principles, techniques, pro-
tocols, and applications of these physical methods of gene delivery and their advan-
tages and limitations, in terms of the kinetics and efficiency of gene delivery, toxicity
profile of delivery, in vivo feasibility of the method, and targeting ability. The com-
parison among various physical, chemical, and biological nonviral systems for DNA
delivery with their advantages and disadvantages has been described in Table 3.1 .
A specific biological or nonbiological method may show enhanced gene expres-
sion, depending on the target cells to be transfected, the force or method applied for
DNA transfer and the specific barriers encountered in vivo , but better transfection may
be achieved by combining different methods of gene transfer. Delivery of a biological
agent by physical or chemical methods has been used to achieve higher gene transfec-
tion and infection, as observed in the expression of proviral SIV genome using a gene
gun at the mucosal surface of a nonhuman primate [8] . By using this technique, a
gene gun is able to deliver a plasmid that expresses a live-attenuated vaccine [9] .
3.2 Naked DNA Delivery
A naked DNA injection, without any carrier, into local tissues or into the systemic
circulation is probably the simplest and safest physical/mechanical approach of
gene delivery. Naked plasmid DNA is an attractive nonviral gene vector because of
its inherent simplicity and because it is easily produced in bacteria and manipulated
using standard recombinant DNA techniques. It shows very little dissemination and
transfection at distant sites following delivery and can be readministered multiple
times into mammals (including primates) without eliciting an immune response.
Also, contrary to common belief, long-term foreign gene expression from naked
plasmid DNA is possible even without chromosome integration if the target cell is
postmitotic (as in muscle) or slowly mitotic (as in hepatocytes) and if an immune
reaction against the foreign protein is not generated [5] .
The first report of direct plasmid expression after direct intramuscular injec-
tion in myofibers was published by Wolff et al. in 1990 [5] . It was an unexpected
finding, where the use of naked nucleic acids was kept as the control for experi-
ments designed to assess the ability of cationic lipids to mediate expression in vivo.