Polymer-based non-viral gene delivery as a concept for the treatment of cancer

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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Gene delivery as a concept for treatment of cancer��

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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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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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Polymer-based non-viral gene delivery as a concept for the treatment of cancer