I.
Introduction
Gene therapy is perceived as a ground-breaking technology with the
promise to cure almost any disease, provided that we understand the genetic and
molecular basis of the malady being treated. However, enthusiasm has rapidly
abated as multiple clinical trials have failed to show efficacy. The limiting
factor seems to be the lack of a suitable delivery system to carry the
therapeutic genes safely and efficiently to the target tissue (Louise, 2006). Gene-transfer technology is still in a
nascent stage owing to several inherent limitations in the existing delivery
methods. While lipid-based vectors (liposomes) provide high transfection
efficiency, their large scale production, reproducibility and cytotoxicity remain
a major concern (Lv et al, 2006). On the other hand, cationic polymers are
robust and relatively biocompatible, but they suffer from poor gene-transfer
efficiency (Pack et al, 2005). Adenoviruses are
the vehicles of choice for cancer gene therapy at this point particularly due
to their ability to overcome the intracellular barriers and the enormous
possibility for recombinant engineering. However, non-specific binding to all
cells that over-express coxsackievirus and adenovirus receptor (CAR), potential
immunogenicity, high costs of production, and the fact that the majority of
cancer cells do not express CAR has limited their use for cancer gene therapy (Thomas et al, 2003; Shen and Nemunaitis, 2006). What
has been long desired is a technology which combines the biocompatibility,
efficiency and the ability to engineer an effective gene-transfer technology.
Since internalization of both viral and non-viral vectors is the first step in
their transfection pathway, knowledge and understanding of their entry
mechanisms is of major importance for the design of efficient viral and
non-viral vehicles for cancer gene therapy.
II. Strengths and
weaknesses of current vectors
A. Viral vectors for
systemic cancer gene therapy
Viruses have evolved to efficiently infect their host, overcome the
cellular barriers and transfer their genetic material into the cellŐs nucleus.
One viral vector that has received considerable attention in cancer gene
therapy is adenovirus. The basic elements of the trafficking pathway for adenovirus
include high affinity binding of the capsid to receptors on the cell surface,
internalization by endocytosis, lysis of the endosomal membrane resulting in
escape to the cytosol, trafficking along microtubules, binding to the nuclear
envelope, and insertion of the viral genome through the nuclear pore (Leopold and Crystal, 2007).
Adenoviruses have high affinity for the CAR and use it to enter the
cells. Although they are highly efficient in transducing cells that
over-express CAR on their surface, they are considered poor gene delivery
systems in cells that have low expression of CAR (Li
et al, 1999). In addition, CAR is expressed on many normal cells which
undermines the ability of this vector to specifically reach target cancer cells
when administered systemically. Thus, adenovirus is not considered a universal
efficient vehicle for cancer gene therapy as the majority of cancer cells do
not over-express CAR (Shen and Nemunaitis, 2006).
Another virus, Herpes Simplex Virus overcomes this deficiency by utilizing a
different receptor to enter cancer cells. The initial attachment of HSV
involves the interaction of viral envelope glycoproteins with the
glycosaminoglycan moieties of cell surface heparan sulfates (Spear et al, 1992).
However, like CAR, expression of heparin sulfates is not unique to cancer cells
and can be found routinely in normal cells. As a result, systemic
administration of HSV could also be problematic.
Attachment of a targeting ligand to the viral capsid has been used as a
means to make adenovirus specifically bind cancer cells and internalize via
receptor mediated endocytosis. One example is attachment of the ligand,
fibroblast growth factor 2 (FGF2) which has affinity for the basic fibroblast
growth factor receptor (FGFR) (Green et al, 2008)
(Figure 1). This receptor
is over-expressed in subpopulations of lung, prostate and breast cancer (Chandler et al, 1999). While promising, the
attachment of the ligand to the virus capsid involves chemical conjugation
during which a significant portion of viruses could become inactive. As a
result, obtaining high titers of active virus for delivery becomes expensive.
Alternatively, retargeted viruses can be genetically engineered through the
abrogation of CAR binding (e.g., Y477A mutation in adenoviral fiber protein)
and insertion of a receptor-specific binding peptide in the HI loop of the
fiber protein (Piao et al, 2009). In this
approach, no chemical conjugation step is involved. However, one potential
problem with this approach is that targeting peptides with considerable 3D
structure could interfere with the proper packaging of the viral capsid
proteins and result in reduced transduction efficiency. Furthermore, such
alterations in receptor targeting could impact transduction efficiency of viruses
due to the change in trafficking routes and internalization pathways (Varga et al, 2000).
B. Are
viral vectors highly immunogenic?
There are five main classes of viral vectors which can be categorized
into two groups (Table 1) according to whether their genomes
integrate into host cellular chromatin (oncoretroviruses and lentiviruses) or
persist in the cell nucleus predominantly as extrachromosomal episomes (AAVs,
adenoviruses and herpes viruses).
Figure 1. Schematic representation of cell transfection by adenoviruses
(Ad). While CAR represents coxsackie adenovirus receptor, FGFR represents
fibroblast growth factor receptor (FGFR).
Out
of these five, only herpes simplex virus (HSV) and adenovirus (Ad) have been
shown to be highly immunogenic. In general, introduction of any non-self
molecule, including viruses, into the body has the potential to trigger an
immune response. However, the level of immune response to the foreign entity is
dependent on the dose, the structure and any previous exposures. For example, a
patient (Jesse Gelsinger) who suffered from a partial deficiency of ornithine
transcarbamylase (OTC) took part in a gene therapy clinical trial conducted at
the University of Pennsylvania in 1999. OTC is a liver enzyme that is required
for the safe removal of excessive nitrogen from amino acids and proteins.
Gelsinger received the highest dose of vector in the trial (3.8 × 1013
particles). After 4 hours of treatment Gelsinger developed a high fever and
within four days of treatment he died from multiorgan failure. A female patient
who received a similar dose (3.6 × 1013 particles) experienced
no unexpected side effects. It has been speculated that previous exposure to a
wild-type virus infection might have sensitized GelsingerŐs immune system to
the vector (Bostanci, 2002). If a lowered dose
of the adenovirus was administered, GelsingerŐs symptoms may not have been as
catastrophic. Therefore, drawing a firm conclusion that viral vectors are
highly immunogenic and deadly is premature.
C. Are
non-viral vectors biocompatible?
Polymeric or liposomal based non-viral vectors are utilized to complex
plasmid DNA forming stable nanoparticles. This complexation protects the DNA
from serum endonucleases and also condenses the DNA into nanosize particles
suitable for cellular uptake. Non-viral polymeric vectors are generally
believed to be non-immunogenic mostly due to their lack of structural
hierarchy. Although there has been reports on the toxicity of such vectors
(e.g., PEI or liposomes) (Lv, Zhang et al, 2006),
in general they are assumed to have low immunogenic potential. Polymers such as
poly (ethylene glycol), for example, have been utilized to sterically stabilize
the surface of particles reducing the interaction of particles with the
elements of the immune system (Chekhonin et al, 2005).
However, two separate groups recently reported that repeated injection of
PEGylated liposomes in rats and mice elicits PEG-specific IgM/IgG (Ishida et al, 2006; Judge et al, 2006). These studies
highlight the potential that even a presumably safe polymer such as PEG can
invoke an immune response if injected in high doses and repeatedly. This in
turn may undermine the ability of PEG to be used as surface stabilizer in drug
delivery systems that need multiple injections to achieve significant
therapeutic response. As a result, drawing a general conclusion that non-viral
vectors are less immunogenic than viral vectors is also premature at this
point. Therefore, there is a continuing need for the development of more
biocompatible and bio-interactive polymers that can reduce immunogenicity. This
in turn enhances blood circulation time of drug delivery systems maximizing
their therapeutic efficacy at the target site.
D. Are
viral vectors more efficient than non-viral vectors?
1. Viral vectors versus targeted non-viral vectors
From the available literature, it is apparent that the efficiency of
non-viral vectors is usually compared with the adenoviral vector which arguably
is the most efficient viral vector (Thomas et al, 2003).
As a result of this comparison, it is generally believed that non-viral vectors
are less efficient. This comparison may not be completely reliable in all
situations as adenoviral vectors are targeted systems which utilize abundant
CAR receptors to enter the cells (Wickham et al. 1993).
When CAR receptors are not abundant the transfection levels are markedly
decreased (Li et al, 1999). Targeted non-viral
vectors are usually equipped with ligands that are intended to bind to
over-expressed receptors. These include growth factor receptors (e.g., FGFR and
HER2) and transferrin. The abundance of these receptors on the surface of the
cells and their affinities towards their corresponding ligands may not be as
high as CAR. Therefore, non-viral vectors that could be as efficient as
adenoviruses in trasfecting dividing cells will show less efficiency when internalizing
through receptors because of the difference in receptor number and binding
affinity. The question then is how viral and targeted non-viral vectors can be
fairly compared in terms of gene transfer efficiency. One potential solution
would be evaluation of transfection efficiency normalized to the abundance of
the receptor being utilized. This is to remove the bias associated with the
receptor numbers. Another answer could be as simple as comparison of FGF2
targeted non-viral vector with FGF2 retargeted adenovirus. In this approach,
the bias associated with receptor binding affinity and internalization pathway
can be eliminated. Alternatively, adenovirus can be compared with non-viral
vectors that are equipped with CAR ligands to target cells. In this way the
bias associated with the number of entry gates as well as receptor binding
affinity will be eliminated. It is also noteworthy that the number of viral or
non-viral particles delivered needs to be kept equal to achieve the same
multiplicity of infection (MOI). To date no study has been reported that has
considered the abovementioned factors in order to appropriately compare viral
versus targeted non-viral vectors.
2. Viral vectors versus non-targeted non-viral
vectors
For non-targeted non-viral vectors, the surface charge of the
nanoparticles usually dictates the binding efficiency to the surface of the
cells. Once complexed with pDNA, the nanoparticles are formulated to have a
slight positive surface charge (e.g., 10-40 mV). This facilitates binding to
the negatively charged phosphate groups on the surface of the cell membranes
resulting in internalization via caveolae or clathrin mediated endocytosis (Midoux et al, 2008). In this scenario, comparison of
viral with non-targeted non-viral vectors would not be appropriate as they
utilize entirely different internalization pathways. Transfection efficiency,
in this case, will be dependent on the cell type not the vector. In one cell
line (e.g., CAR positive), the viral vector will be more efficient than the
non-viral vector, while in another cell line (e.g., CAR negative), the
non-viral vector will show higher efficiency. Therefore, drawing any conclusion
regarding the efficiency of viral vectors versus non-targeted non-viral vector
may not be appropriate.
III. Emerging new technologies
In recent years there has been a great deal of interest on the
development of recombinant polymers (biopolymers) with applications in tissue
engineering, drug delivery and gene therapy (Dreher et
al, 2006; Furgeson et al, 2006; Hatefi et al, 2006, 2007; Canine et al, 2008;
Nettles et al, 2008). The major advantage of the polymers that are
genetically engineered versus chemical synthetic methods is the homogeneity,
control over sterotacticity and full control over the architecture (Urry, 1997). These biopolymers bear the potential to
hybridize the strengths of both viral and non-viral vectors in order to
overcome the extra- and intracellular barriers to efficient, safe and
cost-effective gene delivery. This is due to their versatility, flexibility,
unlimited capacity and most importantly ability to bioengineer at the molecular
level.
In addition to genetically engineered polymers with well-defined
architecture, synthetic inorganic gene carriers (e.g., nano- rods and tubes)
are exciting, emerging technologies that would allow precise control of
composition, size and multifunctionality of the delivery system (Krajcik et al, 2008; Liu et al, 2008). For example,
LeongŐs group recently reported a non-viral gene-delivery system based on
multi-segment bimetallic nanorods with the ability to simultaneously bind
condensed plasmid DNA and targeting ligands in a spatially defined manner (Salem et al, 2003). Although promising, there are
some concerns related to the toxicity and pharmacological fate of inorganic
nanocarriers (Lacerda et al, 2006).
Nonetheless, synthetic inorganic gene carriers have great potential to make a
significant impact on the science of cancer gene therapy.
IV. Conclusion
Lack of an efficient, non-toxic and non-immunogenic gene delivery system
remains the major limiting factor to advancements in cancer gene therapy.
Adenovirus while efficient in some cell lines (CAR positive) raises concerns
about safety as well as targetability. Non-viral vectors while potentially less
immunogenic than viral vectors have not been studied thoroughly enough to
reliably state that they do not trigger major immune responses. Further studies
need to be done in terms of long term administration, dose scheduling, and
treatment thresholds to examine these effects. The efficiency of non-viral
vectors also needs to be reinvestigated taking into account the model system
being used before blanket comparisons between non-viral and viral efficiency
levels can be made. In both non-specific viral and non-viral vectors the use of
targeting ligands is an attractive alternative to non-specific delivery
particularly in cancer therapy. No matter which system, viral or non-viral,
improvements in current technologies continue to be needed.
Acknowledgement
This work was supported in part by the NIH
biotechnology training fellowship (GM008336) to Canine and Reeves
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Arash Hatefi