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ISSN 0582-9879                                          ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(4): 311-316                                    CN 31-1300/Q

　

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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Received: November 18, 2002Accepted: January 6, 2003

This study was supported by the Major Programs of the Chinese Academy of Sciences during the 9th Five-Year Plan Period (No. KSCX2-3-06), the National Natural Science Foundation of China (No. 30120160823) and the National High Technology R&D Program of China  (863 Program) (No. 2001AA217031)

*Corresponding author:  Tel, 86-21-54921126; Fax, 86-21-54921126; e-mail, [email protected]