The goal of gene therapy is to introduce a therapeutic nucleic acid material to cure
genetic deficiencies and a large number of acute diseases. Exciting results from recent
clinical trials demonstrate without doubt the promise of gene therapy. Despite the
high gene transfer efficiency of viral vectors, there are still some drawbacks in
their use due to their immunogenicity and mutagenesis features. Therefore, there is
still room for non-viral methods to be developed since they are safer. However, gene
delivery by non-viral methods is still a major challenge nowadays. The major limiting
factor remains the lack of a suitable and efficient vector for gene delivery.
The challenge is to deliver the nucleic acid to the right intracellular compartment.
Many efforts have been undertaken to identify the cellular barriers that have to be
passed for this issue ([Fig. 1]). First, the nucleic acid has to be protected from nucleases in the extracellular
compartment. Then the plasma membrane has to be crossed. There are two ways to enter
in the cells; either a direct transfer in the cytosol through destabilization of the
plasma membrane or via endocytosis process. This latter is the main way when chemicals
delivery systems are used as carriers. Once internalized, nucleic acids particles
end up inside endosomes where they must escape to reach the cytosol, where mRNA and
siRNA or oligonucleotides can be translated and find their targets, respectively.
In the case of plasmid DNA (pDNA), it must be imported into the nucleus where the
expression machinery takes place. The size of pDNA limits its cytosolic motion and
passive diffusion through pores of nuclear envelope.([Fig. 1]).
Fig. 1 Main barriers for gene transfer by chemical vector: The first barrier is the plasma
membrane. The main route of the DNA / vector complexes is the endocytosis, therefore
the endosomal escape is the second barrier otherwise complexes would reach the lysosomes
and can be degraded. Then, to be expressed the plasmid DNA has to be imported into
the nucleus, this requires a cytosolic diffusion. The main barrier is the nuclear
envelope which is highly selective since it does not allow the passive entry of molecules
having a diameter size more than ~9 nm. Such molecules are able to be imported by
active diffusion which involves the activity of nuclear import machinery via the recognition
of specific nuclear localization signal (NLS).
In chemical based-methods, several strategies have been developed to bypass these
limitations. Protection and stabilization of nucleic acids in extracellular medium
have been achieved by nucleic acid condensation or encapsulation by chemical vectors.
For the endosomal escape, strategies that exploit the proton-sponge effect enabling
endosomal membrane destabilization have been proposed [1]. The nuclear importation of nucleic acid has also been improved by addition of devices
bearing nuclear localization signal which can be put either on the chemical vector
or the nucleic acid. Despite of all of these tremendous efforts, the efficiency of
these chemical based-strategies is still far from that of the viral vectors.
In parallel, physical based methods for gene delivery have been developed. Electric,
magnetic, light or ultrasound fields have been exploited as physical trigger. Among
them, electrotransfer is one of the most used and efficient method but its invasiveness
still hampers its wide application.
Twenty-five years ago, an alternative method for drug delivery based on ultrasound
stimulation was proposed [2]. This method was proven to be more effective when coupled with gaseous microbubbles
[3]. These micron-sized structures containing gas encapsulated by elastic shell have
allowed the improvement of ultrasound imaging. It has been found that microbubbles
oscillations under ultrasound stimulation resulted in an increased permeability of
surrounding cells. The increased uptake by ultrasound has been attributed to the formation
of transient pores on the plasma membrane with a phenomenon called sonoporation which
is amplified when microbubbles are present.
Several studies have been conducted these last years to delineate mechanisms involved
in sonoporation [4],[5]. However, it is still ill-known how exactly cells that are subjected to ultrasound
and microbubbles internalize extracellular compounds, and which cellular responses
ultrasound and microbubble evoke. It was also suggested recently that besides transient
pore formation, endocytosis mechanism might also be involved in the uptake during
ultrasound-mediated drug / gene delivery [6],[7]. The mechanisms of sonoporation are summarized in [Fig. 2]. These results open a new research area. Indeed, the type of mechanism(s) involved
in the delivery could be both dependent on the microbubble chemical composition, the
type of drug to deliver and on the type of insonified tissue or cells. Improving the
knowledge on both extracellular and intracellular fates of microbubbles and their
cargo will be crucial to clearly specify limitations of this method. ([Fig. 2])
Fig. 2 Gene delivery by sonoporation: When activated by ultrasound waves, microbubbles oscillate
and are able to create transient pores in the plasma membrane. Through these pores
can transit small molecules like calcium and hydrogen peroxide that are known to induce
the endocytosis process. So far, it has not been proven if pDNA could be transferred
inside these pores. During sonoporation, pDNA has been found to enter via endocytosis
process. Therefore, it has to face the same barriers as those pointed on during gene
transfer by chemical delivery systems. Microbubbles bearing / loading pDNA must have
specific attributes that would permit pDNA delivery inside the cytosol.
Ultrasound enables control of both the drug release by activating the microbubbles
and the delivery location by positioning the ultrasound probe in a specific area on
the skin. The combination of the ultrasound trigger effect with targeted gas microbubbles
as drug or gene carrier holds a great promise by offering a targeting method controlling
both pDNA release and the location gene expression. The non-invasiveness of this system
renders it superior to other physical methods as electroporation. Still, some challenges
must be overcome to ensure its efficiency.
Ultrasound and microbubbles assisted-gene delivery have been reported to be efficient
in different tissue types including cardiac, endothelium, liver, kidney, tumour, skeletal
muscles, bones and tendons (for a review see [8]). The best result is obtained with 1 MHz of frequency, the other optimal acoustic
parameters clearly being dependent on the tissue type. In most studies reported so
far, the microbubbles type used are those that have been developed as ultrasound contrast
agents. In those reports, experiments have been done by separately injecting microbubbles
and pDNA, mostly after local injection. These bubbles are not able to load and to
complex nucleic acids. Liposomes bubbles, specifically designed for gene transfer,
seem to be effective on a variety of tissue either or not in a targeted form [9].
For systemic injection, the pDNA must be complexed with or loaded in the microbubble
without affecting the acoustic properties of the latter. This is still challenging
even though the production of microbubbles carrying genetic materials via viral particles,
PEI or complexed cationic lipids [10,]
[11] have been produced. However, the gene transfer levels still have to be improved.
It is obvious that designing an efficient microbubble for ultrasound gene delivery
is the next step to further improve this method. Ideally, this microbubble should
be able to interact specifically with its cell target, carry the gene to deliver and
be optimized for nucleic acid delivery to permit its specific expression. Before developing
new bubbles, it would be worthwhile to pinpoint all requirements by clearly delineating
the DNA / microbubble intracellular routing. We have shown that under a specific ultrasound
exposure microbubbles could enter into cells [12]. It is important to note that at these settings an efficient gene delivery was achieved
suggesting that microbubble entry and gene transfer could be linked.
For most genetic deficiencies, treating the patient via intravenous administration
could be more advantageous for gene therapy. In this case, the nucleic acids must
cross the endothelium barrier and reach target tissues without being degraded. Some
studies have also shown that ultrasound used at a specific regime was able to permeabilize
endothelial barriers [13],[14]. For this particular issue, it would be of interest to establish specific ultrasound
settings that could act first on the vascular barrier and then controlling the microbubble
activation leading to gene transfer.
Cells exposed to ultrasound are known to elicit a variety of biological response that
can be deleterious or with therapeutic potential [15]. Recently, Furusawa and colleagues have shown that ultrasound could induce host
DNA modifications up to double breaks [16]. One can ask if it is possible to fine tune the setting to act on the accessibility
of the DNA structure, thus allowing more efficient transgene integration. This is
not as idealistic as it sounds since ultrasound stimulation has been shown to induce
an over-expression of some genes, especially in musculoskeletal tissues which can
sense external stimuli. Acquiring more knowledge on the cellular effects in terms
of molecular signaling will be worthwhile to take advantage of these phenomena for
gene delivery purposes.
Chantal Pichon, Anthony Delalande Center of Molecular Biophysics, University of Orléans
and CNRS, Orléans, France
