Physical methods for gene transfer: Improving the kinetics of gene delivery into cells
Introduction
With recent advances in molecular biology and the sequencing of the human genome, gene therapy is expected to assume a pivotal role in the treatment of genetic diseases. This innovative therapy involves the introduction of healthy copies of mutated or absent genes into target cells so as to promote the expression of normal protein and to restore correct cellular function. The development of gene therapy vectors with sufficient targeting ability, transfection efficiency, and safety must be achieved before gene therapy can be routinely used in man.
The ideal vector for gene delivery would have at least the following characteristics: (i) specificity for the targeted cells; (ii) resistance to metabolic degradation and/or attack by the immune system; (iii) safety, i.e., minimal side effects; and (iv) an ability to express, in an appropriately regulated fashion, the therapeutic gene for as long as required. In general terms, gene delivery methods can be sub-divided into two categories: (a) the use of biological vectors and (b) techniques employing either chemical or physical approaches. The first implicates viral-mediated processes referred to as infection. Retroviruses and adenoviruses are the most commonly used vectors and have already been tested in clinical trials. They offer several advantages, but also many undesired side effects, such as viral toxicity, host immune rejection, as well as being difficult to prepare [1], [2]. Non-viral gene transfer, or transfection, involves treatment of cells by chemical or physical means. Chemical methods cover an array of complexes between DNA and diverse polycations (“polyplexes”) or cationic lipids (“lipoplexes”). Technically, the approach is relatively straightforward and easily scaled-up, and it does not provoke specific immune responses. However, efficiency and targeting remain extremely poor.
A naked DNA injection, without any carrier, into local tissues or into the systemic circulation is probably the simplest and safest ‘physical/mechanical’ approach. However, due to rapid degradation by nucleases and fast clearance by the mononuclear phagocyte system, the expression level, and the area of tissue treated, after a naked DNA injection are severely limited [3], [4]. Consequently, attention has turned to a number of other so-called ‘physical’ manipulations to improve the efficiency (rate and extent) of gene delivery. These methods have also attracted interest for their potential ability to circumvent various “barriers,” which significantly compromise the efficiency of gene delivery, including massive dilution of DNA upon injection, accessibility of the target site, and entry into the cell and the nucleus.
In this review, the following physical methods for gene delivery are discussed: microinjection and particle bombardment (gene gun); electroporation, sonoporation, and laser irradiation; and magnetofection. After a brief description of each technique, their applicability to the enhancement of gene transfer, particularly with respect to the rate and extent of delivery, will be compared to other, especially non-viral, transfection techniques. Finally, we will discuss the advantages and limitations of these physical methods, in terms of the kinetics and efficiency of gene delivery, the toxicity, in vivo feasibility, and targeting ability.
Section snippets
Microinjection
The most direct method to introduce DNA into cells is microinjection, either into the cytoplasm or into the nucleus. This is a microsurgical procedure that is conducted on a single cell, using a glass needle (i.e., a fine, glass, microcapillary pipette), a precision positioning device (a micromanipulator) to control the movement of the micropipette, and a microinjector. Extrusion of fluid containing the genetic material through the micropipette uses hydrostatic pressure. Injections are
Electroporation
A common physical tool to introduce DNA into cells is an electric field. This technique, termed electroporation or electropermeabilization, exposes the cell membrane to high-intensity electrical pulses that can cause transient and localized destabilization of the barrier. During this perturbation, the cell membrane becomes highly permeable to exogenous molecules, such as DNA, present in the surrounding medium. Permeabilization requires that the externally applied electric field surpasses a
Magnetic field-enhanced transfection—magnetofection
Magnetofection is a new method to enhance the introduction of gene vectors into cells [72]. The idea is to associate magnetic nanoparticles with DNA and either its transfection reagent or its virus vector. The magnetic nanoparticles are made of iron oxide, which is biodegradable, with a polymer coating. Their association with the gene vectors is achieved by salt-induced colloidal aggregation. The magnetic particles are then concentrated preferentially into the target cells by the influence of
Kinetics of gene delivery by physical methods
A key advantage of the physical techniques described above is that direct delivery of genetic material into cells can be achieved. This distinguishes these methods clearly from other non-viral gene transfer technologies, such as lipofection or polyfection. That is, gene delivery from a synthetic vector, following cell attachment, involves endocytosis and entrapment into endocytic vesicles, maturation of endosomes into lysosomes, escape from vesicular compartments, migration toward the nucleus
Transfection efficiency
Microinjection is by far the most efficient approach. Up to 100% of the recipient cells may be transfected, and stable, transformed cell lines can be isolated with a frequency of 20–30% after intracellular injection of DNA [100]. The quantity of DNA successfully introduced into each cell is known, thereby reducing waste of expensive plasmid, and transgene expression is easily controlled. It is also possible, for applications requiring a high level of expression, to inject solutions in which the
The future—improving the performance of physical techniques
Enhancing the performance of physical methods for gene delivery translates into the need to increase, primarily, the extent of DNA transfer to the cell nucleus while minimizing “collateral” damage to the tissue being treated. Most of the methods described are able to provoke the relatively rapid opening of the plasma membrane barrier, and permit plasmid to enter into the cell. The challenge, at this point, then becomes how to move the DNA from the cytoplasmic compartment into the nucleus as
Conclusions
Although many advances have been achieved in the field of gene transfer, the major obstacles, poor efficiency and lack of specificity in vivo, remain. Physical methods for gene transfer are relatively new arrivals on the scene, but have demonstrated their potential to directly transfer (and even target) DNA into cells and achieve rapid expression of the transgene. The techniques described in this review have both positive and negative features, in terms of efficiency and practicality, and it is
Acknowledgements
We thank Bracco Research S.A. for the financial support, and Drs.Thierry Bettinger, Feng Yan and Eric Allémann for stimulating discussions.
References (136)
- et al.
Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs
Blood
(2001) High efficiency transformation by direct microinjection of DNA into cultured mammalian cells
Cell
(1980)- et al.
Gene transfer into mouse embryos: production of transgenic mice by pronuclear injection
Methods Enzymol.
(1983) - et al.
Comparison of the CD8+ T cell responses and antitumor effects generated by DNA vaccine administered through gene gun, biojector, and syringe
Vaccine
(2003) - et al.
Optimizing the biolistic process for different biological applications
Methods Enzymol.
(1993) - et al.
Introduction of DNA into rat liver with a hand-held gene gun: distribution of the expressed enzyme, [32P]DNA, and Ca2+ flux
Biochem. Biophys. Res. Commun.
(1997) - et al.
In vivo gene gun-mediated DNA delivery into rodent brain tissue
Biochem. Biophys. Res. Commun.
(2000) - et al.
In vivo transfection of lamprey brain neurons by gene gun delivery of DNA
Exp. Neurol.
(2001) Structure and dynamics of electric field-induced membrane pores as revealed by rapid-freezing electron microscopy
- et al.
In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA
Biochim. Biophys. Acta
(1991)