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https://www.abbs.info ISSN 0582-9879 |
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Mini Review |
Targeting Strategies in
Cancer Gene Therapy
WANG Jin-Hui, LIU Xin-Yuan*
(
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological
Sciences,
the Chinese Academy of Sciences, Shanghai 200031, China )
Abstract Targeting to the tumor tissues can improve the therapeutic
effect of gene transfer by preventing damage of healthy tissues and decreasing
the risk of germ line transduction. Although targeting seems not important for
intratumoral gene delivery, it becomes crucial when systemic gene transfer is
performed. Targeted gene therapy of malignancies can be achieved through
targeted gene delivery or targeted gene transcription. Recent advances in
targeted delivery include the successful use of bifunctional crosslinkers to
target adenoviral and retroviral vectors, inserting short targeting peptides
and larger polypeptide-binding domains into the coat proteins of a number of
different viral vectors, and replication-competent vectors which have been
shown to be promise as anti-cancer agents. Some other non-viral therapeutic
agents, including receptor-mediated DNA or liposome-DNA complex, and bacteria
vehicles have also been developed. Some of these delivery systems are currently
in clinical trials. For targeted and regulable gene transcription, tissue or
tumor specific promoters and some manual regulatory systems are used to
regulate therapeutic gene expression. Antisense oligonucleotides, some ribozyme
and DNAzyme molecules are developed to inactivate genes that are essential to
the development of many tumors.
Key words
targeting; gene therapy; cancer
Cancer gene
therapy represents one of the most rapidly developing areas in pre-clinical and
clinical cancer research. However, some problems need to be solved before this
strategy becomes routinely adopted in clinic. Two of the most important
problems are lack of selectivity of the existing vectors and low efficiency of
gene transfer. Namely, the development of DNA vectors with maximal efficiency
and minimal toxicity is critical for the success of gene therapy and apt to be
accepted by patients and clinicians. It is envisaged that an ideal vector
should be highly targeting, protected from degradation and immune attack, and
safe for the recipient and the environment. It could be administered by
intravenous infusion or injection, whereupon it would concentrate in the organs
or tissues harboring the target cells. Local accumulation of the vector in the
vicinity of the target cells could result in an efficient and selective gene
delivery into the target cells but not into neighboring nontarget cells. The
therapeutic effect of the gene should be targeted and last as long as required
in an appropriately regulated fashion. In recent years, there have been
intensive efforts to generate targetable, injectable vectors based on a variety
of viral and non-viral gene delivery systems.
In this article,
we will review the recent development of strategies used to achieve targeting.
1 Targeted gene delivery
Some progresses
have been made in vector targeting with viral (non-replicative and
replication-competent), non-viral vectors, and some bacterial modalities as
targeted vehicles.
1.1 Targeted
non-replicative viral vectors
1.1.1 Conjugate-based
targeting strategies In
this strategy, a bifunctional bridging agents recognizing both the virus and
the specific cell surface molecule interacts with viral vector and directs the
vector to the targeted cell population.
Heregulin (HRG)
receptors are attractive targets for retroviral vector-based gene delivery
since they are often overexpressed on the surfaces of cancer cells. It was
reported that a TVA-herbeta1 bridge protein, consisting of the extracellular
domain of the TVA receptor for subgroup A avian sarcoma and leukosis virus
(ASLV) and the EGF-like region of HRG β1, could target ASLV-A retroviral vector to cells that express HRG
receptors [1]. Adenovirus can also be targeted using anti-knob
antibodies chemically conjugated to different ligands recognizing specific cell
surface receptors. For example, Adenovirus was targeted to cancer cells
expressing the c-erbB-2/HER-2/neu oncogene by a bispecific
adapter protein, sCARfC6.5 which consisted of soluble coxsackie virus and
adenovirus receptor (sCAR), phage T4 fibritin polypeptide, and C6.5
single-chain fragment variable (scFv) against c-erbB-2 oncoprotein[2].
Other bifunctional crosslinkers were also generated targeting other cell
surface receptors such as fibroblast growth factor receptor (FGFR)[3]
and epidermal growth factor receptor (EGFR)[4].
A limitation
with the bifunctional crosslinker approach is that the neutralizing antibody
fragment is not covalently linked to the vector particle. Therefore, the
complex might dissociate in the blood stream following intravenous
administration. Moreover, infection efficiencies are relatively low with this
approach[5 ] .
1.1.2 Genetic
targeting strategies Different
viruses (and their derivative vectors) utilize different strategies to
introduce their nucleic acid into the target cells. There are several factors
that influence the transfer efficiency of viral vectors: some unmodified
viruses can not infect human cells because the cells lack the required
receptors; the binding of some viruses and their receptors can not result in an
effective infection; some viruses infect human cells promiscuously. Genetically
modified strategies are explored to change the viruses’ targets according to
the problems described above.
For viruses
without required receptors on cells, they are modified so that they can bind to
a receptor that is selectively expressed on the surface of the targeted cell
population. There have been numerous attempts to retarget ecotropic murine
leukemia virus (MLV)-based vectors. Incorporation of a ligand into these
chimeric MLV envelopes will enhance the infectivity of these vectors in some
cases, depending on the receptor that is targeted. For example, the approach
was successful for EGFR[6].
Viruses are
modified to enhance their ability to interact with a particular subset of
target cells without increasing their infectivity on nontarget cells. The
enhancement of gene delivery by adenovirus vectors was achieved on certain
types (vascular endothelium and smooth muscle cells, which express
heparan-containing cell surface receptors) of target cells when the viral fiber
protein was modified by the addition of a poly-lysine tail[7] or an
integrin-binding RGD (Arg-Gly-Asp) peptide into the viral capsid[8].
Other strategies
are developed to restrict the virus infectivity on particular subsets of human
target cells. Such restriction needs to block the interaction between virus and
its natural receptor and redirect the viral particle to a receptor expressed
only on the target cells. This has been achieved by mutating/deleting the
domain required for receptor binding and adding new binding domains potentially
to retarget these vectors. In this regard, fiberless adenoviral particles[9]
and MLVs with mutations in the receptor-binding domain[10] had been
generated. High affinity growth factor receptor-binding domains have been
displayed on the N-terminus of retroviral envelope glycoproteins or C-terminal
of adenovirus knob. Infectivity of the modified vector incorporating an
envelope on which EGF was displayed was restricted on EGF receptor-positive
cells but not on negative ones[11]. One new strategy is to block
virus infectivity by grafting a protease-cleavable receptor-blocking domain
onto the viral coat protein. In the target tissue where there is a high local
activity of the relevant protease, the site that anchors the blocking domain to
viral surface is cleaved and virus infectivity is restored. This had been
demonstrated for cell-surface proteases[12] and for soluble
proteases[13].
Genetic
modification can form a single-component system and has advantages over
conjugate-based strategy by its high transfer efficiency.
1.2 Targeted replication-competent viral
vectors
Use of
non-replicative viruses will have a limited efficiency of gene transfer. In
contrast, replicative viral vectors will allow genes to be delivered initially
to a small number of tumor cells to replicate and then to be transferred to
neighboring cells as the infection spreads, which can significantly increase
the efficacy of gene delivery. Obviously， it is critical for this type of strategy that the replication cycle
of the replication-competent vector should be targeted to the tumor cells or to
the tumor micro-environment. Three major strategies come forth. (1) Completely
or partially delete viral genes that become dispensable in tumor cells, such as
the genes responsible for activation the cell cycle through p53 or Rb binding.
E1B gene deleted adenoviruses that replicate preferentially in p53-deficient
target cells have been developed for the treatment of head and neck tumors. The
therapeutic effect reached 63%[14]. Replication-competent herpes
simplex virus vectors that are unable to make ribonucleotide reductase had been
shown to replicate selectively in rapidly dividing cell populations and not in
normal tissues[15]. (2) Control transcription of viral genes by
replacing the native viral promoters with tumor-specific[16] or
hypoxia-regulated promoters[17]. (3) Interfere with the signaling
pathway in tumor cells. Reovirus had been shown to replicate selectively in
cells carrying ras-activating mutations [18] and vesicular
stomatitis virus (VSV) was a replication-competent virus for treatment of
interferon non-responsive tumors[19].
Replicative viruses can be used as single agents, vectors, or in
combination with chemotherapy. Incorporation of therapeutic genes into the
replication-competent viral vectors represents a promising method to improve
its efficacy/toxicity ratio.
1.3 Targeted non-viral delivery systems
Compared to
viral vectors, non-viral systems are particularly suitable for gene therapy
with respect to simplicity of use, lack of immune response, ease of large-scale
production and DNA packaging. In spite of its clinically well tolerance, the
overall transfection efficiency of naked DNA or liposome-DNA complexes in
vivo remains low. Ligands or binding domains to surface receptors can be
used to improve its transfection efficiency by direct binding to DNA (polyplex)
or coupled to the liposome-DNA complex delivery system (lipoplex).
Ligands to surface receptors included asialoglycoprotein, basic fibroblast
growth factor, transferrin, and adhesion molecules. Peptides containing an
RGD motif with high affinity for
integrins and a short polylysine segment for electrostatic binding of DNA could
efficiently transfer genetic material to different cell types[20].
Efficient transfection was demonstrated in a number of cell types in vitro[21]
and in bronchial and
alveolar cells in vivo, with transgene expression sustaining for at
least three to seven days[22]. Nonviral delivery methods, when
combined with a noninvasive, clinically applicable imaging assay, were shown to
aid greatly in the optimization of gene therapy approaches for cancer [23]
.
1.4 Targeted
bacteria vehicles
Other targeted live vehicles such as
bacteria have also been investigated for cancer gene therapy. Bacteria have
large genome size, readily express multiple therapeutic transgenes, and their
proliferation can be controlled with antibiotics.
Two anaerobic bacteria have been developed as gene transfer vehicles, Clostridium
and Bifidobacterium, Gram-positive non-pathogenic anaerobes selectively
germinate and replicate in necrotic and hypoxic regions of solid tumors[20],
which makes them a promising tumor-selective vehicle for gene therapeutics. In
vivo, intravenous (i.v.) injection of spores of cytosine deaminase
(CD)-transfected C. sporogens followed by systemic administration of the
prodrug 5-fluorocytosine(5-FC) induced significant antitumor activity [24].
Attenuated hyperinvasive auxotrophic mutants of Salmonella typhimurium
have often more than 1000 times higher specificity for tumors than for any
other tissue. Auxotrophic mutations make these bacteria highly safe and form
the basis for maintaining tumor specificity [25]. When these
auxotrophs were inoculated intraperitoneally into melanoma bearing mice, they
suppressed tumor growth and prolonged average survival to twice that of
untreated mice. Moreover, when the animals were inoculated with Salmonella
expressing the gene of herpes simplex virus thymidine kinase (HSV-TK),
ganciclovir(GCV)-mediated, dose-dependent suppression of tumor growth was
observed [26] . Compared to Salmonella, the lack of
pathogenesis of Bifidobacterium might be advantageous when used in human
therapeutic treatment.
2 Targeted gene
transcription
Targeted gene delivery has limitations in
its ability to specifically target tumor cells, for receptors some strategies
adopted are not unique in the target cells. So specific and regulable
expression of the therapeutic gene to tumor cells or targeted inactivation of
specific genes responsible for tumor development is explored. There has been
spectacular progress in this field of study in the past 10 years and
transcriptional targeting has been shown highly feasible in the context of most
viral or non-viral vectors.
2.1 Targeted therapeutic gene
transcription
Currently, a variety of therapeutic genes
such as suicide genes, antiangiogenic genes, tumor-suppressor genes and
cytokine genes are used in the field of cancer gene therapy. To construct
transcriptionally targeted vectors for the therapeutic genes, several
strategies are used: many tissue specific transcriptional regulatory sequences
have been developed, such as flt-1 promoter of the epithelial cells and glial
fibrillary acidic protein promoter of glioma cells; tumor specific
transcriptional regulatory sequences can be employed, such as CEA
promoter preferentially activated in adenocarcinoma cells, AFP promoter in
hepatocellular carcinoma cells and human telomerase reverse transcriptase (hTERT)
promoter in 85%－90% tumor
cells; promoters activated by pathological or physical conditions are used,
such as use of hypoxia-responsive elements to achieve hypoxia-regulated gene
expression[27] and use of ionizing radiation regulated egr-1
gene promoter to achieve radiation induced gene expression [28]; to
fine-tune the gene expression in a regulated fashion, some manual regulatory
systems have been developed based on the regulation mechanism of gene
expression, such as tet-on and tet-off systems that regulate
therapeutic gene expression by administration of oral drug tetracycline, and
RU486 system that regulates gene expression by activation of the GAL4 chimeric
transactivator with administration of oral drug RU486[29] .
One limitation of cell type-specific promoter is that the transgene
expression is influenced by promoter activity in cancer cells. Cre/loxP system
can be used to overcome this problem. It had been recently applied in
CEA-producing tumors therapy[30]. A pair of recombinant adenoviruses
was constructed. One expressed the Cre recombinase (Cre) gene under the control
of the CEA promoter (Ad.CEA-Cre). The other contained the HSV-TK
gene separated from the strong CAG promoter by insertion of the neomycin
resistance (neo) gene (Ad.lox-TK). The HSV-TK gene of the latter
Ad was designed to be activated through excisional deletion of the neo
gene by Cre enzyme released from the former one only when CEA-producing cells
were infected simultaneously with these Ads. Coinfection by these Ads rendered
a human CEA-producing cancer cell line 8.4-fold more sensitive to GCV compared
with infection by Ad.CEA-TK alone, the HSV-TK gene of which was directly
regulated by the CEA promoter. Intratumoral injection of Ad.CEA-Cre combined
with Ad.lox-TK followed by GCV treatment almost completely eradicated
CEA-producing tumors established in the subcutis of athymic mice.
2.2 Targeted pathogenic gene
inactivation
Molecular basis of a range of malignancies
are activation, mutations, or alterations in the expression of oncogenes and
tumor-suppressor genes. Antisense strategies have been developed to target
mRNAs of pathogenic genes. Antisense oligonucleotides offer the possibility of
specific, rational, genetic-based therapeutics. However, the results from
animal models and clinical trials have often been disappointing, with little
evidence of clinical benefit from the antisense treatment[31].
Recently, ribozyme and DNAzyme molecules
have been developed for targeting cancer gene therapy. Ribozymes are RNA
molecules that lead to site-specific cleavage of a target mRNA in a catalytic
manner. The appeal of ribozyme technology as a potential cancer therapy lies in
the possibility of designing a site-specific ribozyme for virtually any target
that the sequence is known, or its role in disease pathogenesis has been
proven. Ribozymes are viewed as more potent therapeutic agents, because they have
a number of advantages over antisense molecules. (1) The cleavage action of
ribozymes permanently inactivates the target gene. (2) The catalytic nature of
ribozyme molecules implies that, in theory, lower concentrations would be
required, because one ribozyme may act on a number of target molecules. The
preformed secondary structure of ribozymes also imparts a target accessibility
advantage over long antisense molecules, the inherent secondary structure of
which can prevent binding to the complementary RNA. (3) Ribozymes might be
expected to exhibit greater target specificity because gene inactivation
requires binding of the complementary flanking arms and the presence of a
specific cleavage consensus sequence[32] .
DNAzymes are single-stranded
oligodeo-xynucleotide with enzymatic activity. The typical DNAzyme, known as
the “10-23” model, is capable of cleaving
single-stranded RNA at specific sites under simulated physiological conditions.
DNAzymes offer several significant advantages compared with ribozyme molecules,
including easier synthesis and decreased sensitivity to chemical or enzymatic
degradation. Furthermore, DNAzymes exhibit greater substrate flexibility
compared with conventional and hammerhead ribozymes. The 10-23 DNAzymes can
cleave effectively any unpaired purine and pyrimidine of mRNA transcripts. As a
result, DNAzymes can be designed specifically to recognize the AU nucleotides
of the start codon. Because the translation start site and its neighboring
bases have little secondary structure, DNAzymes often reduce their substrate
mRNA levels without significant amounts of screening. These characteristics of
DNAzymes make them promising candidates for in vivo targeted
oligonucleotide therapy. DNAzyme targeting vascular endothelial growth factor
receptor 2 (VEGFR2) had been developed to inhibit tumor angiogenesis[33].
Marked cell death in the peripheral regions of the tumor accompanied by a
reduction in blood vessel density was observed after DNAzyme administration in
breast tumor bearing mice.
Transcriptional targeting in no way
obviates the need for targetable, injectable vectors. It does, however, provide
a very reassuring safety net to protect normal tissues from collateral damage
that can occur when toxic transgenes are inaccurately delivered. Dual targeting
strategy was explored in adenoviral vectors at both transduction and
transcription level [34]. A higher degree of specificity for cancer
cells has been achieved by combining the complementary approaches of
transductional and transcriptional targeting, each of which is imperfect or “leaky” by itself .
3 Conclusion
Gene therapy is a promising but also
difficult approach. After 13 years since the approval of the first clinical
trial, it is still in the early stages of development. The development of
targetable and injectable vector will determine the success of a number of
different gene therapy systems. In this aspect, the field of targeting is
advancing rapidly. To date, major clinical experience has been gained with
viral vectors, but the biosafety of the modification is still disputable. The
effective replication-competent virus, when combined with target gene
transcription system, represents an excellent approach for cancer gene therapy,
but raises serious safety concerns relating to the inherent mutagenicity of
animal viruses, so do the tumor-specific bacteria that may induce pathogenesis and
the induced inflammation in humans. These problems need to be solved in future
clinical studies. Nevertheless, in the field of gene therapy, the data
collected so far are encouraging, and illustrate both feasibility and future
promise for cancer treatment. At present, the key part that needs to be
fulfilled in this progress is targeting.
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