polymer-based non-viral gene delivery as a concept for the treatment of cancer
TRANSCRIPT
Review
Polymer-based non-viral gene delivery
as a concept for the treatment of cancer
Anna Halama1,3, Micha³ Kuliñski1,4, Tadeusz Librowski2,
Stanis³aw Lochyñski1
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Abstract:
Gene therapy has become a promising technique for the treatment of cancer. Nevertheless, the success of gene therapy depends on
the effectiveness of the vector. The challenge of a gene carrier is to deliver exogenous DNA from the site of administration into the
nucleus of the appropriate target cell. Polymer-based vectors are biologically safe, have low production costs and are efficient tools
for gene therapy. Although non-degradable polyplexes exhibit high gene expression levels, their application potential is limited due
to their inability to be effectively eliminated, which results in cytotoxicity. The development of biodegradable polymers has allowed
for high levels of transfection without cytotoxicity. For site-specific targeting of polyplexes, further modifications, such as incorpo-
ration of ligands, can be performed. Most expectations have been addressed to polyplexes architecture according it dynamic re-
sponse with the microenvironment.
Key words:
cancer, polymers, gene delivery, synthetic vectors, polyplexes
Introduction
Gene therapy is a technique used in molecular medi-
cine that aims to cure a genetic disease by providing
the patient with a correct copy of the defective gene.
In contrast, traditional drug-based approaches treat
the pathological result but do not repair the underly-
ing genetic defect [2, 38]. Human genetic diseases are
the result of gene mutations or deletions that lead to
attenuation of normal metabolic pathways [4], dys-
regulation of the cell cycle [41] or ligand/receptor
dysfunction. Diseases that can be treated with gene
therapy can be divided into genetic and acquired. The
idea of adding the correct gene sequence to mutated
cells via gene delivery has been shown to be plausible
and can potentially lead to elimination of the disease [1].
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Cancer cells are resistant to apoptosis and continue
to multiply in an unregulated manner. This dysregula-
tion is due to mutations in DNA, overexpression of
particular genes that encode proteins that promote cel-
lular growth or decreased expression of genes that en-
code proteins that control and inhibit cell growth [11].
Conventional cancer treatments, such as surgery and
radio- or chemotherapy, are effectively used against
certain types of cancers [3, 33, 42]. However, these
therapeutic approaches have multiple drawbacks, in-
cluding limited specificity, the occurrence of non-
specific side effects and risk of recurrence due to in-
complete eradication of cancer cells. These limita-
tions demonstrate the importance of developing alter-
native therapies that are effective, specific and have
minimal side effects [43]. Gene therapy is an emerg-
ing and developing field in the treatment of cancer.
However, such a therapeutic approach is only effec-
tive if the genes of interest are specifically and suc-
cessfully delivered to the target cells.
Implementation of transgene delivery
into target cells
Therapeutic genes can be delivered into target cells by
viral or synthetic vectors. The optimal gene carriers
must be easy to prepare at a low cost and should allow
for unrestricted nucleic acid payloads. In addition,
a delivery vector must maintain the stability of the
formed polyplex of nucleic acids and must deliver the
genetic cargo to the target site with high specificity.
Furthermore, repeated administration of the vector
must have minimal toxicity with efficient gene ex-
pression in the target cells [8].
The synthetic vectors currently available are less
efficient for gene delivery in comparison to viruses
with slightly lower levels of gene expression. How-
ever, viral vectors are limited in their transgene load-
ing size, production costs and their inherent toxicity
profile, which leads to inflammation and immunogen-
icity. In addition, the death of a patient treated with
adenoviral vector gene therapy has raised serious pub-
lic concerns about the safety of viral vectors [21].
Synthetic vectors, in contrast, have low immunogen-
icity, higher biosafety, considerably lower production
costs and can easily undergo chemical modifications.
These characteristics make synthetic vectors attrac-
tive alternatives for gene therapy.
Pathways and barriers for the production
of synthetic vectors
Synthetic gene carrier systems can be formulated from
both nucleic acids (pDNA or siRNA) and cationic lipids
to form lipoplexes or from nucleic acids and cationic
polymers to form polyplexes. The positively charged
moieties of these synthetic vectors can interact with nega-
tively charged phosphate groups on DNA, resulting in the
formation of positively charged complexes (Fig. 1A).
The condensation of DNA with polycations or cati-
onic lipids protects it from degradation by nucleases.
The complexes are then attracted via electrostatic in-
teractions to the negatively charged cell surface,
which consists of molecules such as proteoglycan
[30]. Uptake of the complexes proceeds by adsorptive
endocytosis, pinocytosis or phagocytosis. It is important
to note that the positively charged poly- or lipoplexes
may interact unspecifically with non-target sites. How-
ever, this problem can be partially solved by coupling
ligands to the transgene carriers, enabling the com-
plex to target specific cells and minimizing interac-
tions with unwanted cells.
The interaction of the complexes with and their up-
take by target cells are the first barriers that need to be
overcome. Next, the complexes need to be released
from the endosome and must migrate towards the nu-
cleus. The DNA released intracellularly then needs to
penetrate across the nuclear membrane to get inside
the nucleus where it can be transcribed (Fig. 1B). Sev-
eral additional barriers, which must also be overcome,
are encountered when the complexes are applied in
vivo, either locally or systemically. When the com-
plexes are applied intratumorally, they have to diffuse
through the solid tumor mass. Hence, the complexes
can interact with extracellular matrix components or
with necrotic cellular material, reducing their trans-
fection efficiency [30].
This local application scheme is limited for some
tumor types, such as melanoma. For these types of tu-
mors, systemic delivery of genes may be more effi-
cient. However, in this case, complexes must over-
come the additional barrier of being transported
through the blood circulation [36]. Particle size is
a crucial parameter that can influence successful de-
livery of the complexes, especially for long-term cir-
culation in the blood. Additionally, complexes must
not interact with blood components and should re-
main stable under physiological conditions.
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Polymers as synthetic vectors
As shown in Figure 1A, polycationic polymers con-
dense negatively charged DNA via electrostatic inter-
actions, forming polyplexes that can deliver transge-
nes to the target cells. Polymers can be promising car-
riers for gene therapy when the particles used result in
high transfection efficiency with minimal toxicity. In
addition, under optimal conditions, polyplexes may
also prolong the expression of therapeutic genes, im-
proving the therapeutic effects of treatment. The de-
mand for the production of an optimal synthetic vec-
tor has resulted in the development of several poly-
mers. In general, these polymers can be divided into
non-degradable and degradable polymers.
Non-degradable polymers
The most common polymer used for DNA delivery is
polyethylenimine (PEI, 22 kDa), which is water-
soluble and consists of multiple ethylenamine units.
The high density of positive charge in this polymer is
contributed by the amino groups. In vitro, the gene
expression levels of PEI are comparable to those of
viral vectors [14]. This expression efficiency is asso-
ciated with the high cationic charge of the polyplexes,
which gives them a high affinity for negatively
charged cells. In addition, the buffering capability of
PEI allows for the efficient endosomal release of the
complexes after uptake. The amino groups can act
like “proton sponges” inside the endosome, leading to
an osmotic swallow effect followed by breaking of
the endosomal membrane [6, 13]. Moreover, the di-
rect interaction of positively charged PEI particles
with the negatively charged internal endosomal mem-
brane may also play a role in membrane destruction
[5, 6, 23]. PEI is available in linear (as LPEI, 22 kDa)
and branched (as BPEI, 25 kDa) forms. Both forms
are widely used because they provide high levels of
gene expression.
PEI-based DNA particle systems were recently
tested in humans for the treatment of bladder cancer.
The results revealed that there was extensive tumor
regression after intravesical vector application in
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treated patients. In 2004, Ohana et al. [31] reported
the use of PEI as a carrier for a construct expressing
diphtheria toxin A (DTA) driven by the H19 promoter
(oncofetal gene commonly expressed in various tu-
mor tissues) in a preliminary clinical trial that enlisted
two patients. The patients were treated intravesically
with 2 mg of the PEI/toxin vector DTA-H19 complex
once a week for 6 weeks. Video imaging showed a re-
duction in tumor size of more than 75% [31]. These
results emphasize the increased necessity of develop-
ing non-viral vectors. However, specific delivery of
nucleic acids to the tumor site after systemic admini-
stration remains a major challenge in the optimization
of gene vector formulations [37].
It is important to note that one major drawback of
synthetic vectors is their toxicity profile, as their high
positive charge causes their aggregation and unspe-
cific reaction with blood components and erythro-
cytes in vivo [7, 29]. In addition, PEI cannot be prop-
erly eliminated from or metabolized by the body.
These features can lead to accumulation of the com-
plexes within organs and, as a consequence, increase
their toxicity. This drawback of non-degradable poly-
mers has led to the development of biodegradable
polymers that can overcome cytotoxicity but still
maintain high transfection levels.
Degradable polymers
Degradable polymers have recently become attractive
alternative vectors for gene delivery. Their ability to
be degraded results in lower toxicity and makes them
easier to be eliminated from the body. Several reports
have described the use of biodegradable polymers, in-
cluding poly(L-lysine) (PLL) [18] and their analogs
poly(�-[4-aminobutyl]-L-glycolic acid (PAGA) [44,
48] and poly(4-hydroxy)-L-proline ester [16]. The ar-
chitecture of these polymers is comprised mainly of
hydrolysis-sensitive bonds, such as esters, acetals or
hydrazones, which can be broken down by enzymatic
or chemical degradation leading to their decomposi-
tion into smaller metabolites. Although degradable,
low molecular weight molecules, such as PLL (LMW
PLL), exhibit low or no cytotoxicity when compared
to high molecular weight molecules (HMW), they
show relatively low transfection efficiency. The reason
for this can be explained by the fast hydrolyzation of
LMW PLL and its rapid removal from the blood cir-
culation, leading to poor transfection activity in vivo
[19, 20]. However, further improvements in the archi-
tecture of these biodegradable polymers are important
for overcoming the toxic effects associated with other
vectors.
Optimization
Currently, all synthetic gene carriers exhibit compara-
ble effectiveness, in terms of transgene expression, to
viral vectors under in vivo conditions. Viruses are ca-
pable of overcoming all biological delivery barriers.
Thus, their architecture performs different functions
for the efficient delivery of genes due to changes trig-
gered by the environment. Viruses are generally as-
sembled in a controlled fashion but undergo uncon-
trolled disassembly upon infection of neighboring
cells. The ability of using polymer-based synthetic
vectors opens the possibility to develop “smart artifi-
cial viruses”. One advantage of using these smart arti-
ficial virus particles for gene therapy is the ease with
which one can change their static functional construc-
tion to a more multifunctional one. Two crucial fac-
tors for the improvement of these vectors are increas-
ing their colloidal stability under physiological condi-
tions, which is important during the delivery phase, as
well as increasing their affinity for the target cells [39].
Stabilization of polyplexes
The effectiveness of synthetic polyplexes appears to
strongly correlate with their colloidal stability. Since
proteins and polyanions can slowly penetrate the
polymer layer of the complexes destabilizing the core
of the vector, several research groups have concen-
trated on enhancing transfection efficiency by im-
proving the stability of polyplexes. One of the strate-
gies taken for this purpose has been to modify the sur-
face of the vector through cross-linking. In this
approach, the cross-linking agent forms a net-like sys-
tem on the surface of the polyplexes, holding the
DNA inside and consequently decreasing the possibil-
ity of DNA release.
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The stabilization of polyplexes can be achieved ei-
ther through pre- or post-cross-linking. Pre-cross-
linking modifications occur prior to polyplexes forma-
tion; this method has been previously performed by
McKenzie et al. [22]. In this procedure, cross-linked pep-
tides are developed and polymerized through degradable
disulfide bond formation, which results in the formation
of a structure that condenses DNA in a high, stable man-
ner, allowing for efficient in vitro gene transfer.
Oupicky et al. [32] and Neu et al. [26–28] de-
scribed the stabilization of polyplexes after their for-
mation, a process termed post-cross-linking. This
method also results in improved stability of the com-
plexes, leading to high gene expression levels. How-
ever, it is important that complexes are stabilized
enough to overcome biological conditions without
compromising their ability to release the DNA cargo
within the target cells.
“Smart” polyplexes – receptor mediated
gene transfer
Proper delivery of exogenous DNA from the site of
administration into the nucleus of the appropriate tar-
get cells is a challenge for all gene carriers. Receptor-
mediated delivery of molecules is a cellular process
by which ligands bind to specific cell surface recep-
tors, resulting in subsequent internalization of the re-
ceptor/ligand complexes by the cell [25]. In order for
polyplexes to be targeted specifically to the desired
site, the incorporation of ligands has to be introduced.
In addition, the architecture of the polyplex should re-
spond in a dynamic manner to the microenvironment.
Previous reports have shown that improving the
specificity of polyplexes toward their target increases
their transfection efficiency. Wu and colleagues dem-
onstrated that asialoorosomucoid-polylysine-ligated
DNA complexes could be specifically targeted to the
asialoglycoprotein receptor on hepatocytes [35, 45,
46]. There are many other ligands that can be coupled
to polyplexes, such as insulin, EGF and others.
Another common ligand, first described by Wagner
and colleagues [40], is transferrin (Tf). Due to their
fast growth rate, cancer cells have a greater require-
ment for iron, resulting in their increased expression
of transferrin receptor [12]. Transferrin is an iron-
binding protein expressed on the cell membrane; upon
binding to its appropriate receptor, transferrin can be
internalized and recycled by the cell. Kircheis et al.
[15] described a dual function for transferrin. The large
size of transferrin allows it to act as a shield for com-
plexes and also decreases their surface charge. This ef-
fect results in a decrease in non-specific interactions
with other molecules, such as blood components, and
an increase in tumor specificity. In addition, the high
specificity of Tf-targeted vectors for cancer cells in-
creases their transfection efficiency [17, 29, 40].
Recently Davis et al. [9] reported the use of a cy-
clodextrin (CD)-based construct for use as an siRNA
carrier in cancer clinical trials. These polyplexes were
stabilized with PEG-adamantane groups and modified
with transferrin. The siRNA used in this case targeted
RRM2 and led to a significant anti-proliferative effect
on cancer cells (human, mouse, rat and monkey). In
preclinical studies, non-human primates were treated
intravenously with a construct containing 1 mg/ml of
siRNA. After multiple injections, no toxicity was de-
tected [10]. Currently CD-siRNA complexes are be-
ing evaluated in Phase I clinical trials for the treat-
ment of solid tumors [24].
Future challenges for the use of polymer-
based vectors in gene therapy
The main goal of gene therapy is to efficiently deliver
therapeutic genes to target cells and to achieve their
controlled, high expression levels with low toxicity.
In comparison to viral vectors, synthetic vectors are
attractive gene carriers for use in gene therapy be-
cause of their biosafety and ease of being chemically
modified.
While polymer-based non-viral gene delivery is
a promising method for the treatment of cancer, the
drawback of this method lies in the difficulty of effec-
tively introducing the genetic cargo into the target
cells. Further studies will be aimed at finding ways to
improve the structure of the polymers through chemi-
cal modification in order to overcome this drawback.
The use of polymers as gene carriers has already
been tested in clinical trials. Seymour et al. [34] re-
ported the use of the hydrophilic polymer poly[N-(2-
hydroxypropyl)methacrylamide] (pHPMA) as a car-
rier for cytotoxic drugs in Phase I and II clinical trials
for the treatment of lung and liver cancer patients at
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Gene delivery as a concept for treatment of cancer���� ������ �� ��
Queen Elizabeth Hospital. They describe that, “The
polymer is very versatile and can easily be used for
covalent coating of the surface of the polyplexes. In
fact, results using this approach have been excellent”.
Such an optimized polymer structure provides the
possibility for both active and passive targeting of tu-
mors and also allows for other disease sites within the
body to be targeted.
The stabilization of a therapeutic transgene carrier
requires a balance between stability in the circulation
and its expected reduction following entry into cells.
Such a structure will allow for efficient transcription
of the genetic cargo following cell entry. In addition,
incorporation of one or more ligands that target spe-
cific receptors into the structure of the polyplexes can
improve their specificity for cancer cells by increas-
ing their uptake by receptors.
Messenger RNA (mRNA), rather than pDNA, seems
to be an attractive alternative for non-viral gene delivery.
By using mRNA, the nuclear membrane, which is
a major obstacle for delivery of pDNA, can be
avoided because mRNA exerts its effects in the cyto-
plasm. In addition, the risk of insertional mutagenesis
can be avoided. Moreover, the use of mRNA results in
high gene expression levels in non-dividing and post-
mitotic cells, an effect which is extremely difficult
when using pDNA [47].
Synthetic vectors are still insufficient to achieve
high gene expression levels effective enough for use
in gene therapy. However, improving polyplexes to
make them capable of overcoming both extracellular
and intracellular barriers could be a powerful system
for use in clinical trials.
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