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British Medical Bulletin 64:201-225 (2002)
© 2002 The British Council

Gene therapy for colorectal cancer

Daniel H Palmer*, Ming-Jen Chen* and David J Kerr{dagger}

* CRUK Institute for Cancer Studies, The Medical School, University of Birmingham, Birmingham, UK
{dagger} Department of Clinical Pharmacology, Radcliffe Infirmary, Oxford, UK


   Abstract
Top
 Abstract
Introduction
 Gene therapy trials for...
 Challenges
 Conclusions and key points...
 References
 
Gene therapy has been developed as a potential novel treatment modality for colorectal cancer. The preclinical data have been promising and several clinical trials are under way for colorectal cancer. Data from many phase 1 trials have proven the safety of the reagents, but have not yet demonstrated significant therapeutic benefit. In order to refine this approach, continuing efforts should be made to improve the antitumour potency, efficiency of gene delivery, and accuracy of gene targeting. It is likely that gene therapy will be integrated into pre-existing therapies including surgery, chemotherapy and radiotherapy to establish its niche in tomorrow's medicine.


   Introduction
Top
 Abstract
 Introduction
Gene therapy trials for...
 Challenges
 Conclusions and key points...
 References
 
With the advances in biotechnology, gene therapy has been explored as a possible modality of cancer treatment. To date, over 600 gene therapy clinical trial protocols have been activated in the US, 60% of which pertain to cancer gene therapy. Nearly 3500 patients have been treated within these protocols, of which about 2400 were patients with cancer1. Similarly in the UK, of approximately 70 gene therapy protocols approved or under review by GTAC, 70% relate to cancer gene therapy2. The majority of these trials have been phase I dose finding/safety/toxicity studies, with less than 1% being phase III randomised studies against current best practice.

The theoretical advantage of gene therapy approaches for cancer treatment is the specific targeting of the tumour site, hence reducing unwanted systemic toxicity that is often a dose-limiting factor of conventional drug therapy. This advantage relies on extremely well-regulated mechanisms that control the accuracy of gene delivery and expression at the specific targeted site(s). Both prerequisites are in fact obstacles to its success. Indeed, there has been little clinical efficacy shown in the many phase I clinical trials. Nevertheless, the efforts in developing novel gene delivery vectors, either viral or non-viral, in discovering new therapeutic genes and in exploring tumour biology in order to overcome the hurdles, are currently making this conceptually new field of medicine potentially promising. This article aims to provide an overview of the current state of gene therapy, especially focusing on the current clinical trials, for colorectal cancer.


   Gene therapy trials for colorectal cancer
Top
 Abstract
 Introduction
 Gene therapy trials for...
Challenges
 Conclusions and key points...
 References
 
Immune stimulation

The aim of immune stimulation (immunogene therapy) is to activate a systemic and tumour-specific immune response, which may be either cell-mediated or antibody-dependent, against the tumour cells. The attraction of this approach is specificity of systemic targeting and the potential for amplification of the effect once precisely activated. On the other hand, the difficulties of this approach arise from the fact that, during tumour progression, many cancer cells are able to evade host immune surveillance by down-regulating immunogenic mechanisms, such as the decreased expression of major histocompatibility complex (MHC) molecules or co-stimulatory molecules, the secretion of immuno-inhibitory cytokines, induction of T-cell anergy, etc. Furthermore, the continually dynamic evolution of tumour antigens over time adds more complexity to immune stimulation approaches that target specific tumour antigen(s)3,4.

To overcome these hurdles, different strategies have been explored which stimulate the host immune system to recognize tumour antigen(s) and hence generate endogenous antitumour activity, for example, a tumour-specific cytotoxic T-cell immune response. Essentially, the activation of tumour-specific T cells requires three synergistic signals, including the presentation of tumour antigen by antigen presenting cells (APC) to T-helper cells; the interaction between co-stimulatory factors (e.g. B7.1) and CD28 ligand; and the secretion of cytokines by activated T-helper cell (e.g. interleukin-2 [IL-2]). When antigen is presented simultaneously with the co-stimulatory molecule B7.1, there is an increase in IL-2 secretion, increased expression of IL-2 receptor and increased T-cell proliferation (Fig. 1). Several approaches to stimulate these key factors have been tested in preclinical experiments and have now entered clinical trials, including the following.



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  		Fig. 1 Cell-mediated immunity against tumours was proposed as crucial in antitumour immunity. Tumour antigens are taken up and processed by antigen presenting cells (APC), and presented to class II receptors on T-helper cells. An effective response requires the interaction between co-stimulatory signal, B7 and CD28 ligand, causing T-helper cell activation. As a consequence, cytokines (e.g. IL-2) were secreted from helper cells to activate tumour-specific cytotoxic lymphocytes to recognise tumour cells via class I receptors and kill tumour cells.

 
Utilisation of human leukocyte antigen (HLA) to stimulate T-cell response
 HLA class I molecules are down-regulated in up to 60% of colorectal cancers. Animal studies have demonstrated that the expression of foreign MHC (the analogue of HLA in humans) on tumours can induce a T-cell dependent anti-tumour immune response, not only to the foreign MHC but also to previously unrecognised tumour associated antigens5.

On the basis of preclinical models, gene transfer of HLA-B7 has been examined in clinical trials. In one trial, an allogeneic HLA-B7 plasmid in a lipid vector was administered via direct intratumoural injection to HLA-B7-negative patients with melanoma. This treatment was well tolerated with only minor toxicity. Gene transfer rate was 93% when measured by polymerase chain reaction (PCR) and HLA-B7 protein was found in 50% of biopsied tumours by immunohistochemistry (IHC). Eight of 15 evaluated patients developed anti-HLA-B7 CD8+ cytotoxic T-cells (CTLs), and 7 patients had tumour reduction (4 partial responses)6. A phase II trial reported a response rate in evaluable patients approaching 15%, including two complete responses, demonstrating this to be a safe and active treatment against melanoma7.

This trial has been extended to include patients with hepatic colorectal metastases, in which the vector was injected intratumourally under ultrasound-guidance. Of 15 patients evaluated, 14 had detectable transgene DNA by PCR, and HLA-B7 protein was detected by IHC in 63% of biopsied lesions. A biological response was evident, as induction of B7-specific CTLs in peripheral blood of 8 patients and also infiltration of CD8+ T-cells into some tumours on IHC. However, no objective responses were seen. This may be due to the tumour burden and the large size of the injected lesions. Again treatment was well-tolerated with no evidence of autoimmune induction confirming this to be a feasible, non-toxic treatment, although further studies to assess efficacy are required8.

In summary, HLA-B7 gene transfer has shown the ability to elicit systemic antitumour immune response in humans. Yet, no local antitumour effect was shown in hepatic colorectal metastases as has been demonstrated in melanomas.

Utilization of cytokines to stimulate T-cell response
 Cytokines play a key role in co-ordinating the immune response. Therefore, the insertion of genes encoding cytokines presents a potential strategy to increase the immunogenicity of tumours and overcome immune tolerance. Different cytokines may recruit different inflammatory/immune cells, but the end result is often similar. Pre-clinical models have tested a range of cytokines including IL-2, IL-4 IL-12, granulocyte macrophage-colony stimulating factor (GM-CSF), and interferon-{gamma} (IFN-{gamma}). In general, in vivo models confirm that tumour-specific immunity can be generated by cytokine-transduced tumour cells. However, while this is often strong enough to prevent tumour formation/growth when re-challenged with new, untransduced tumour cells, it is less efficient in eradicating established tumours. Gene therapy with IL-2 has shown antitumour efficacy in a murine model of colorectal cancer and has been investigated in patients with colorectal cancers9.

Ex vivo IL-2 transduction to autologous fibroblasts: In a phase I clinical trial, 10 patients with colorectal cancer were treated with IL-2 gene therapy. Autologous fibroblasts (used for their ease of growth in tissue culture and trasducibility by retrovirus vectors expressing cytokines) were transduced with a retrovirus carrying the IL-2 gene and mixed with autologous irradiated tumour cells prior to subcutaneous re-injection. This treatment was well-tolerated, inducing only mild/moderate fatigue and transient flu-like symptoms. In 2 of 6 evaluable patients, there was successful induction of tumour-specific CTL precursors and in 5 of 10 patients there was evidence of immunological memory by delayed-type hypersensitivity skin reaction to subsequent injection.

This trial shows it is possible to induce a cellular immune response using IL-2 gene therapy in patients with colorectal cancer safely and with minimal toxicity. However, no objective responses were demonstrated10.

Ex vivo IL-4 transduction to autologous fibroblasts: A phase I study has been undertaken in which patients with advanced cancer were vaccinated with a combination of IL-4-transduced fibroblasts plus autologous irradiated tumour cells. Tissue biopsies of vaccination sites revealed that IL-4 mRNA could be detected by RT-PCR even 2 weeks after initial vaccination, confirming the effectiveness of this strategy in generating IL-4 in vivo. However, induction of an antitumour immune response was not reported11.

Ex vivo IL-2 transduction to autologous immune effector cells: Another approach has been to transfect autologous immune effector cells with the IL-2 gene. Preclinical studies have shown that cytokine-induced killer cells, CIKs (non-MHC restricted cytotoxic lymphocytes) can eradicate tumours in nude mice. Further, transfection of CIKs with cytokine genes can increase proliferation and cytotoxicity. In a phase I study, 10 patients (predominantly with colorectal cancer) were treated with autologous CIKs derived from peripheral blood mononuclear cells, PBMCs, transfected ex vivo by electroporation with an IL-2 plasmid before re-infusion intravenously. Toxicity was mild/moderate, comprising grade 2 fever in 3 patients which resolved within 24 h. Transfected cells were detected in blood in patients 2 weeks after infusion. There was evidence of biological activity as indicated by an increase in serum IFN-{gamma}, GM-CSF and TGF-ß during treatment and also an increase in the cytotoxic activity of circulating lymphocytes tested against a range of HLA-matched carcinoma cell lines. Three patients were reported to demonstrate disease stabilisation following treatment. One patient with follicular B-cell lymphoma achieved a complete response12.

IL-2 transduction using other vectors: Two further phase I studies treating patients with a range of advanced cancers have utilized either allogeneic fibroblasts secreting IL-2, or an IL-2 DNA/lipid complex (‘leuvectin’) delivered by direct intratumoural injection13,14. Both approaches were well-tolerated with evidence of biological activity (detection of IL-2 on tumour biopsy and tumour infiltration by T-cells) in vivo as well as clinical objective responses in some patients (with melanoma or renal carcinoma).

In summary, cytokine gene therapy appears to be safe when mediated via a number of different vectors including subcutaneous injection of retrovirus-transduced fibroblasts with autologous tumour cells, cytokine-transduced immune effector cells, and direct intratumoural injection of cationic lipid vectors. Despite evidence of biological activity of these strategies in humans, objective responses have been rare. It is postulated that this disappointing lack of response may be due to down-regulation of MHC as a tumour escape mechanism. So, co-expression of cytokines with, say, HLA-B7 may augment antitumour immune response and may be a useful therapeutic strategy. Meanwhile, preclinical studies using a replication-deficient adenovirus vector to deliver IL-2 suggest that the adenoviral vector itself may enhance CTL recognition of tumour antigens, implying that such a vector may have therapeutic advantage in cytokine-mediated immunotherapy15.

Utilization of carcino-embryonic antigen (CEA) in cancer vaccines
 Tumour-associated antigens have been identified for a range of human tumours including viral antigens (e.g. HPV E6, E7), mutated oncogenes (e.g. ras) and non-mutated onco–fetal proteins (e.g. CEA). Since T-cell epitopes to these antigens have been identified, they may serve as targets for CTLs under appropriate conditions. Molecular characterisation of tumour-associated antigens and identification of their genes has allowed the development of recombinant vaccines in which a vector is used to introduce DNA encoding tumour-associated antigens into patients. Viruses (especially ‘pox viruses’) efficiently present antigen and induce both humoral and cell-mediated responses. Therefore, co-presentation of tumour-associated protein with the vaccinia vector may enhance immunogenicity and increase the possibility of tumour rejection.

CEA is a cell-surface glycoprotein over-expressed on the majority of colorectal cancer cells, and is only expressed at low levels in normal colon and biliary epithelium. On the basis of differential expression levels, CEA has been selected as a potential target for immunotherapy approaches. Different methods have been utilised in clinical trials.

Vaccinia vector: The immunization of CEA transgenic mice with recombinant vaccinia virus containing the CEA gene induced anti-CEA antibodies and cell-mediated immune response against subsequent challenges with CEA-expressing tumours16. Several phase I trials have now tested recombinant vaccinia vectors encoding full-length CEA administered subcutaneously or intradermally at doses between 107–108 PFU to patients with metastatic colorectal cancer. These studies confirm this to be a well-tolerated treatment with toxicity confined to low-grade fever, fatigue and inflammation at the injection site17,18. The vaccine was able to induce a CTL response to CEA epitopes. Anti-CEA antibodies could also be detected and, although these were of low affinity and avidity, it is still the first demonstration that such antibodies could be generated in response to a recombinant vaccinia virus19.

Canary pox vector: A potential problem with vaccinia vectors is the generation of neutralising antibodies, which may limit efficacy. This has lead to the use of canary pox vectors. This virus is not pathogenic in humans and does not replicate in human cells. It may, therefore, be given repeatedly without neutralisation by antibodies. A recombinant canarypox virus containing the human CEA gene has demonstrated antitumour efficacy in mice20. In a phase I trial of this vector (‘Avipox’), patients with advanced CEA-positive tumours were treated with 3-monthly intramuscular injections. In 7 of 9 evaluated patients, CEA-specific CTL responses were induced; however, no objective responses were seen21,22.

Designer T-cell: A novel approach to generating an immune response to CEA is the development of anti-CEA ‘designer T-cells'. In a phase I trial, T-cells from patients were transduced by retrovirus delivery of chimaeric Ig-TCR genes to generate immune-effector cells which bind specifically to CEA-positive cells before re-infusion. This treatment was well-tolerated up to doses of 1011 T-cells. It was reported that there was symptomatic improvement in some patients and in one patient a transient reduction in serum CEA (nadir at day 9, but back to base-line by day 23), although no objective reductions in tumour volume were documented23.

Transfer of CEA and co-stimulatory molecules, B7.1
 Most tumours do not express co-stimulatory molecules and so tumour-associated antigens are not presented efficiently. This may represent one mechanism by which tumours evade immune recognition. Therefore, expression of such molecules on tumours may enable presentation of tumour antigens directly to T-cells, thus obviating the need for CD4+ helper cells or antigen presenting cells (APCs). Further, systemic immunity against unmodified tumour cells (‘distant by-stander effect’) may be evoked. Since T-cell activation requires both a specific antigen epitope and a co-stimulatory signal, a further canary pox vector expressing human CEA and B7.1 has been constructed. A phase I study of 18 patients with CEA-expressing adenocarcinoma to determine the safety and efficacy of this vector showed it to be well-tolerated up to 4.5–108 PFU (the highest dose level) with no autoimmune reactions. Minor side-effects included mild pain at injection site, alternations of liver function test (7 patients), anaemia (1 patient) and thrombocytopenia (3 patients). Two of 13 patients with colorectal cancer achieved stable disease. Importantly, this correlated with an increase in CEA-specific precursor T-cells. These patients underwent further vaccination that augmented this CEA-specific T-cell response. Although this is a small phase I study, it suggests that adding a co-stimulatory molecule to virus-based vaccines against tumour-associated antigens may improve immunological responses24: larger studies are warranted.

Overall, immunogene-therapy approaches are attractive and consist of about two-thirds of the on-going clinical trials for cancer treatment. The clinical trials described here have so far failed to demonstrate clinically significant response despite clear biological activation in the form of antibody- and cell-mediated response. The discrepancy may be due, at least in part, to the dynamic evolution of tumour antigens as a result of negative selection. However, it may also highlight the limitations of traditional clinical trial design whereby efficacy must first be demonstrated in the setting of advanced disease. This may not be appropriate for immunogene therapy, which is likely to be most effective against minimal residual disease. It may then be appropriate to test these therapies in randomised controlled trials in an adjuvant/minimal residual disease setting, perhaps in combination with standard adjuvant therapy such as chemotherapy.

Furthermore, more efficient and potent mechanisms of mediating antigen presentation should be explored. Indeed, some groups have focused on the dendritic cell (DC), a professional antigen-presenting cell, in order to facilitate efficient tumour antigen presentation to the immune system. Dendritic cell-based immunotherapies such as DCs modified to express the co-stimulatory CD40 ligand have been shown to cause tumour regression and led to increased survival in mice with syngeneic CT26 colon cancer xenografts25. In humans, DC-based modalities have been reported to cause remission of cancer in trials of B-cell lymphoma, prostate cancer, and melanoma26. A pilot study using CEA RNA-loaded DCs to stimulate CEA-specific T-cell immunity has been designed to test the safety and immune response in patients with CEA-expressing cancers.

Mutant gene correction

Gene correction, like the correction of monogenetic anomaly, is a logical approach in cancer therapy based on the understanding of the carcinogenic processes at the molecular level. However, for most cancers, including colorectal cancer, this goal is elusive because malignant transformation is usually accompanied by an accumulation of genetic mutations, as well as clonal heterogeneity. Furthermore, the success of this approach very much relies on a high efficiency of gene expression at the tumour site(s) in order to achieve therapeutic benefit. Nevertheless, the phenotypic correction of key genetic aberrations of malignant cells has shown the potential to trigger induction of apoptosis in preclinical studies. Different strategies including tumour suppressor gene correction (e.g. p53) or oncogene suppression (e.g. K-ras) have shown antitumoural effect in animal models of colorectal cancer and p53 gene correction delivered in adenoviral vectors is being tested in clinical trials in combination with conventional chemotherapy.

p53 tumour suppressor gene correction
 About 50% of colorectal cancers harbour p53 mutations. It has been shown in an animal model that re-expression of wild-type p53 in p53-mutated colon cancer xenografts can lead to an inhibition of tumour growth and increased animal survival27. An additional bonus for wild-type p53 correction is its by-stander effect, which is thought to be due to the anti-angiogenic effect in tumours with p53 mutation28. Many clinical trials using a replication-deficient adenoviral vector to deliver wild-type p53 to a range of human tumours have been carried out. Initial studies demonstrated the safety of direct intratumoural injection of these vectors and confirmed p53 gene expression even in the presence of an anti-adenovirus immune response.

A phase I study has assessed the safety and efficacy of a single dose of adenovirus-delivered p53 (SCH58500) administered via the hepatic artery to patients with hepatic colorectal metastases with the aim of maximising tumour cell exposure and minimising system exposure. Treatment was well-tolerated up to the maximum dose of 2.5–1012 virus particles with toxicity comprising grade 1/2 fever, flu-like symptoms and in 4 out of 16 patients, a transient asymptomatic rise in liver transaminases. Of 12 patients who went on to receive intrahepatic chemotherapy with 5-fluorodeoxyuridine, 11 achieved a partial response29. This compares to partial response rates of about 50% in historical controls30.

Another group have examined the safety, tolerability and pharmacokinetics of a replication-deficient adenovirus p53 vector (RPR/INGN-201) given intravenously to patients with advanced cancer. Five patients were treated at the maximum dose of 1–1012 virus particles, one of them developed dose-limiting toxicity in the way of grade 3 diarrhoea. Other toxicity was mild, and repeated monthly administration was possible, with one patient with advanced colorectal cancer receiving 10 cycles safely. Data pertaining to intratumoural p53 expression are pending31.

In summary, the phase I trials have proved the safety of p53 gene transfer by adenoviral vectors via intrahepatic arterial or intravenous routes. Further study should focus on the potential synergistic effect between p53 correction and conventional therapies, since preclinical studies suggest that restoration of wild-type p53 can restore chemo- and radiosensitivity of tumours32,33. The low toxicity of adenovirus-delivered p53 should enable this treatment modality to be combined with standard treatments in clinical practice.

Other gene correction strategies
 Many colorectal tumours are positive for microsatellite instability (MSI+). Such tumours commonly have mutation in the retinoblastoma zinc finger gene, RIZ1, but rarely have p53 mutations. In mouse xenograft models, such tumours do not respond to adenovirus-delivered p53 gene therapy whereas adenovirus-delivered RIZ1 can suppress tumour growth and induce apoptosis, suggesting that this may be a useful therapy for MSI+ colorectal cancer34.

Mutation of K-ras is common to many malignancies of the gastrointestinal tract. This provides a potential target for antisense oligonucleotide therapy. The introduction of synthetic oligonucleotides capable of hybridisation to specific complementary mRNAs can block the expression of a single protein that plays a critical role in tumour growth. Preclinical studies of K-ras anti-sense therapy suggest this to be a safe, relatively non-toxic treatment35.

Prodrug activation

Enzyme prodrug systems, also called suicide gene therapy or gene-directed enzyme prodrug system (GDEPT), are alternatives to systemic chemotherapy. This involves gene transfer, for example via a viral vector (virus-directed enzyme prodrug therapy, VDEPT) to express viral, bacterial or fungal enzymes in tumour cells. The enzyme can convert an inactive prodrug into a toxic metabolite, leading to tumour cell death. Compared to systemic chemotherapy, the merit of this approach is the potential of localizing the toxic effects to tumour cells while avoiding toxicity to the normal cells, such as bone marrow and gastrointestinal tract. Yet, the main obstacle of this method is the limited gene transfer efficiency at the tumour site by vectors currently available. However, this hurdle may be partially overcome by the ‘by-stander effect’ (Fig. 2). The by-stander effect refers to extension of the cytotoxicity of the activated drug to non-transduced cells. This may be a local effect mediated by passage of the toxic metabolite (or other apoptotic factor from dying cells) to neighbouring cells either by passive diffusion, via gap junctions or via apoptotic vesicles. Alternatively, there may be an immune-mediated response that could induce a distant by-stander effect. This has been observed in murine GDEPT models in which regression of distant tumour deposits is seen but is reduced in athymic animals36. A logical extension of this phenomenon may be to combine GDEPT with cytokine gene therapy in order to maximise this effect.



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  		Fig. 2 Virus directed enzyme prodrug therapy. Transduced gene encoding enzyme that converts non-toxic prodrug into toxic metabolite, leading to cancer cell death, can be mediated by viral vector. The toxic metabolite can kill adjacent uninfected cancer cells via cell-to-cell contact or released from dead cell, i.e. by-stander effect. Note: arrow means activation.

 
Another concern is that activated prodrugs may actually inhibit the activity of viral vectors, limiting the amount of prodrug activating enzyme that could be produced. Nevertheless, this short-coming could be exploited for good purpose if the viral vector needed to be controlled.

A number of prodrug activation systems are currently being developed.

Herpes simplex virus – thymidine kinase (TK)/ganciclovir
 Phosphorylation of ganciclovir produces the toxic metabolite GCV-triphosphate, which competes with dGTP and inhibits DNA synthesis. This reaction is catalysed by herpes simplex virus (HSV)-TK, 1000-fold more efficiently than the human nucleoside kinase, making this an attractive model for GDEPT. In a syngeneic murine model of colorectal cancer, the HSV-TK/GCV system could achieve complete tumour regression when only 9% of cells express the TK gene. The lipid-insoluble GCV metabolite cannot diffuse into adjacent cells suggesting that the by-stander effect may be mediated by gap-junction transport or via an immune response37. Interestingly, there is evidence of a ‘vaccination effect’ in that, following re-challenge with untransduced tumours, tumour regression can occur.

This system has been shown to be well-tolerated and has achieved clinical responses in phase I trials of brain and prostate cancer. Similar studies have been undertaken using a replication-deficient adenovirus vector to deliver RSV-TK with similar preclinical results. A phase I trial utilising direct intratumoural injection of this vector followed by a fixed dose of GCV in patients with hepatic metastatic colorectal cancer has been undertaken. Sixteen patients were treated with escalating doses of virus up to 1–1013 particles. This treatment was well-tolerated with no dose-limiting toxicity observed, confirming the safety of the adenovirus vector delivered by the intratumoural route38.

Cytosine deaminase/5-fluorocytosine
 Bacterial or fungal cytosine deaminase (CD) is able to convert the antifungal agent, 5-fluorocytosine (5-FC), into 5-fluorouracil (5-FU), one of the most effective chemotherapeutic agents for colorectal cancer. The fungal CD was reported to be superior to its bacterial counterpart. The possible limitation of this system is that 5-FU cytotoxicity is cell-cycle dependent and only a proportion of the tumour cells are in S-phase at a certain time. However, a profound by-stander effect is seen in vivo. Significant tumour regression has been achieved when only 2% of cells in xenografts expressed cytosine deaminase39. Furthermore, the combination of uracil phosphoribosyl transferase (UPRT) with CD has been shown to enhance the antitumoural effect possibly by increasing the turnover of 5-FU to 5-fluorodeoxyuracil monophosphate, thereby accelerating a rate-limiting step in conversion of 5-FU to its cytotoxic metabolite40.

A phase I trial of a replication-deficient adenovirus carrying the Escherichia coli CD gene (Ad-GVCD-10) given intratumourally followed by oral 5-FC to patients with hepatic metastatic colorectal cancer is underway. Dose-escalation to a maximum of 2–109 PFU is planned. The trial comprises of two arms, one of which is treated with vector and prodrug only, the other in which the tumour is removed after treatment so that histological and molecular analysis can be undertaken: results are awaited41.

Nitroreductase/CB 1954
 Nitroreductase can convert the prodrug CB 1954 to a highly toxic bifunctional alkylating agent, which can cause interstrand DNA cross-links, leading to cell death. This effect is cell-cycle independent. In cell-mixing experiments, a significant by-stander effect was seen when only 10% of pancreatic cancer cells expressing nitroreductase were treated with CB 195442.

A phase I dose escalating study of the prodrug CB 1954 has already been completed, establishing the intravenous dose of CB 1954 that can be delivered safely. Pharmacokinetic analysis showed that levels sufficient for clinically significant prodrug activation could be achieved, based on data from preclinical models43. Meanwhile, in the other arm of this on-going phase I study, an E1, E3-deleted adenovirus containing the nitroreductase gene, under the control of a cytomegalovirus promoter, is given to patients with hepatic colorectal metastasis or hepatocellular carcinoma by ultrasound-guided intratumoural injection. CB 1954 will be given intravenously 48 h after viral injection, once an adequate level of nitroreductase gene expression has been detected in resected hepatic tumours from operable patients. So far, preliminary data from this trial have demonstrated safety of intratumoural vector administration up to 1x1011 virus particles. Immunohistochemical analysis of resected tumours has confirmed nitroreductase expression increasing in a dose-dependent manner44.

Generally, the viral vectors used in these trials were delivered by intratumoural injection directly to the tumour site. This approach secures accurate tumour targeting, but may limit use to solitary tumours rather than to systemic disease. Clinical efficacy has not yet been shown.

On-going research on other enzyme-prodrug systems is still in preclinical development including cyclophosphamide/cytochrome P45045; carboxypeptidase G2/CMDA46; and carboxylesterase/irinotecan47.

Further studies to improve antitumour potency are also underway to examine to role of ‘double’ suicide gene therapy in which two enzymes are delivered followed by the administration of the corresponding prodrugs. For example, in vitro studies using a TK/CD fusion gene suggest an additive or even synergistic interaction of this combination when compared to each system independently48. Similarly, enzyme/prodrug systems combined with cytokine gene therapy are being investigated with promising results49.

A further level of specificity for these systems is the insertion of a tumour specific promotor, for example the CEA tumour promoter, to regulate gene expression so that even if normal cells were infected, the enzyme would not be transcribed. It is envisaged that this would allow regional administration of the vector to the liver in patients undergoing resection of a primary colorectal tumour. This would be followed by systemic administration of prodrug. Given the favourable growth kinetics of microscopic metastases following resection of the primary tumour, GDEPT may play a useful role in this adjuvant setting.

Genetically modified oncolytic virus therapy

The concept of using biological agents such as viruses to eliminate cancer first emerged in the early 20th century. The viruses used in the early clinical trials existed naturally and did not show clinical efficacy, probably due to the limited knowledge of virology at that time. In recent years, genetically modified oncolytic viruses, including adenovirus, herpes simplex virus and reovirus, have been developed and tested. These viruses require key tumourigenic pathways to be mutated for viral replication and, hence, can selectively replicate in and lyse tumour cells while sparing normal cells50. Among them, mutants of adenovirus and herpes simplex viruses are already in trials for various cancers.

The development of replication-competent viral vectors may have the potential to improve the disappointingly low transduction rate of therapeutic gene and the relatively low expression in many gene therapy protocols. It is envisaged that conditionally replicating vectors may be able to overcome these hurdles and, further, may have the potential to reach disseminated metastases51.

Oncolytic adenovirus
 The wild-type adenovirus was first used in the treatment of cervical cancer. Recently, an E1-B attenuated adenovirus, dl 1520 (ONYX-015) has been tested on more than 200 patients with head and neck cancer, hepatic colorectal metastasis, ovarian cancer and pancreatic cancer. This mutant virus was engineered not to express the E1B-55-kDa virus protein and, therefore, was initially reported to replicate specifically in cancer cells lacking functional p53, leading to cell lysis52. However, it was subsequently found that this virus could also replicate efficiently in several tumour cell lines with wild-type p53. These contradictory results raised doubt about the specificity of dl 1520-mediated killing effect in p53-mutated cells53. Nevertheless, recent data have showed that the loss of p14-mediated Mdm2 inhibition plays an important role in supporting the replication of this virus in tumour cells with wild-type p5354.

Oncolytic adenoviruses can evoke immune responses including cytokine release, neutralising antibodies, and cell-mediated responses. It is possible that such responses may have a beneficial local antitumour effect. However this response may destroy the infectious virus, thus limiting its direct lytic effect. It should be noted, however, that viral pharmacokinetics remain unchanged between the first cycle (low antibody) and subsequent cycles (higher antibody titres). Further, the immune response may contribute to the side-effects of this treatment modality.

In a phase I/II trial, ONYX-015 was injected via hepatic artery infusion into metastatic hepatic tumours. Two courses of a 5-day infusion of 5-FU/folinic acid were given concurrently. The virus was well-tolerated at the dose of 1011 PFU/infusion without dose-limiting toxicities, although most of the patients developed grade I/II fever and a few patients developed rigors after viral injection. Preliminary data showed partial responses in 2 of the 4 evaluable patients, which is comparable to the standard chemotherapeutic treatment. More patients are still needed to evaluate the results of this study55.

Another trial using the same virus administered via hepatic artery infusion, intravenous infusion or intratumoural injection, without chemotherapy, was conducted in 16 patients with primary or metastatic hepatic tumour (mainly from colorectal primaries). Tumour necrosis after viral injection was seen on CT scanning and histological analysis in all patients. No severe side-effects were observed at a dose of 3–1011 PFU56.

In summary, ONYX-015 has been investigated in several clinical trials treating a range of tumours. Doses up to 2–1012 virus particles have been well-tolerated when administered intratumourally, via the hepatic artery, intraperitoneally and intravenously. No dose-limiting toxicity has been observed. Virus replication has been observed after administration by all routes, but to a variable extent depending on tumour type. There is also confirmation that distant tumours can be infected following systemic delivery. Interestingly, ONYX-015 has demonstrated very little efficacy as a single agent in head and neck cancer (0–14% objective response rate). However, clinical benefit has been seen where combined with chemotherapeutic agents. ONYX-015 in combination with chemotherapy (5-FU and cisplatin) has also showed promising results in a trial of head and neck tumours, with a complete response rate of 27% and a partial response rate of 36%57. The combination of oncolytic virus with chemotherapeutic agents seemed synergistic but randomised, properly powered trials comparing chemotherapy versus chemotherapy plus virus are required.

Recently, a novel E1A-attenuated adenovirus was reported to replicate specifically in tumours with pRb checkpoint impairment. Importantly, a more potent antitumour effect was seen compared to ONYX-015 in preclinical experiments, indicating that more investigation of this field is warranted58

Herpes simplex virus (HSV)
 The original concept that genetically engineered viruses could specifically target tumour cells derived from the observation that deletion of the TK gene from HSV allowed viral replication exclusively in mitotic cells59. The current oncolytic HSVs are engineered with mutations in one or both of two other genes: (i) viral ribonucleotide reductase (ICP6), the loss of which restricts lytic virus replication to dividing cells that retain sufficient ribonucleotide reductase activity to support replication of the virus; and (ii) viral ICP34.4, mutation of which allows continued protein synthesis by blocking the shut-down of host cell protein synthesis normally associated with HSV infection thereby enhancing the generation of virus progeny60,61. Such attenuated HSVs have been engineered and exploited in cancer trials for brain tumours and prostate cancer62,63.

Some variants have been tested for their therapeutic potential against colorectal cancer. One such virus, NV-1020, has shown oncolytic properties in syngeneic mice with a hepatic colon cancer model. This virus is currently in clinical trials for patients with hepatic colorectal cancer metastases64. It is of note that the antitumour efficacy appears to be unaffected by pre-existing immunity to herpes infection65.

A potential advantage of the use of oncolytic HSV is its sensitivity to antiviral agents such as acyclovir, which could be used to control any unwanted infection.

Other oncolytic viruses
 Reovirus is a double-stranded RNA virus with tropism for respiratory and gastrointestinal epithelium. In normal cells, there is a translational blockade of reovirus mRNAs mediated by double-stranded RNA protein kinase, inhibiting lytic virus infection. This block is overcome by ras expression66. So, reovirus can target cells with activated ras signalling pathway, resulting in cell lysis. Since this pathway is constitutively activated in approximately 60% of human colorectal cancers, this virus may have a potential role in gene therapy for this disease.

Oncolytic viruses in combination with other gene therapy strategies
 Replicating viruses have the potential to infect a greater proportion of tumour cells and to increase transgene expression. It is attractive, therefore, to combine these viruses with gene therapy strategies such as prodrug activation or cytokine expression by inserting the appropriate cDNA into the vector (Fig. 3).



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  		Fig. 3 The use of replication-competent viral vector as a carrier for therapeutic gene delivery. The potential advantages are: (i) the dual killing effect caused by viral oncolysis and the therapeutic gene system, for example, enzyme prodrug system; and (ii) the continuing infection by the replicated viruses to surrounding tumor cells which were not infected initially.

 
Preclinical studies using the combination of a replication-competent HSV vector encoding the TK gene have been performed. In a colorectal cancer model, the addition of GCV actually reduced the cytotoxic effect of the oncolytic vector, probably because of the antiviral activity of the activated prodrug67.

More encouraging results have been reported with E1B-attenuated adenovirus vectors encoding a combination of CD and TK68. Data showed that the vector alone was cytotoxic, but the addition of both 5-FU and GCV did further enhance cell killing. Similar results are emerging with the combination of a replicating adenovirus vector and ntr/CB1954 (personal communication). Combinations of replicating vectors encoding cytokines are also showing promise69. Interesting data regarding a potentially synergistic interaction between oncolytic viruses and radiotherapy are also emerging, with evidence that radiotherapy may enhance viral replication within tumours70.

Overall, progress is being made in the clinical development of oncolytic viruses. Future clinical studies will address the optimum combination of replicating vector, gene insert, chemotherapy and radiotherapy to maximise their therapeutic potential.

Myeloprotective gene transfer in conjunction with chemotherapy

Chemotherapeutic treatment for cancer is usually limited by toxicity to normal tissues such as bone marrow. Severe myelotoxicity can lead to compromised immunity and fatal opportunistic infection can then occur. To overcome this problem and to improve the therapeutic index, the transfer of a drug resistance gene to haematopoietic progenitor cells may allow higher doses of chemotherapeutic drugs which can improve disease control while protecting patients from myelotoxicity.

The human MDR1 gene can confer resistance to a wide range of chemotherapeutic drugs including doxorubicin, paclitaxel and etoposide. It is postulated that transfer of the MDR1 gene to haematopoietic stem cells would confer chemoprotection, thereby allowing optimum dose intensity of chemotherapy and minimising morbidity and mortality. Preclinical studies in mice using retrovirus delivery of the MDR1 gene to peripheral blood progenitors ex vivo have confirmed the feasibility of this strategy71. As well as MDR1, a number of other drug resistance genes are being investigated.

Preclinical study with dihydropyrimidine dehydrogenase gene transfer conferred the tested cells increased resistance to 5-FU, a first line drug for colorectal cancer72. Likewise, the transduction of bone marrow progenitor cells with a mutant O6-methylguanine DNA methyltransferase gene (mutant MGMT gene) encoding a mutant O6-alkylguanine-DNA alkyltransferase (AGT), G156A, can provide marrow cells tolerance to the combined use of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) with an AGT inhibitor, O6-benylguanine (BG). Thus, this approach showed the advantage of allowing repeated administrations of BCNU and BG to mice with BCNU-resistant xenografts to achieve tumour growth delay73. A phase I trial using a retroviral vector to transfer mutant MGMT gene to human haematopoietic progenitors is currently under way for patients with solid tumours including colorectal cancer74.

So far, clinical trials have demonstrated the safety of this technique although the number of reconstituting transduced cells and insufficient transgene expression has limited efficacy. Improvements in vector technology and in stem cell isolation from peripheral blood should facilitate progress in this field75.

Antisense oligonucleotide therapy

The antisense approach to cancer gene therapy is based on the understanding of the key genes which, when overexpressed, are responsible for tumour development. In the case of colorectal cancer such genes include MDM2, ß-catenin, Bcl-2 and matrilysin. The Watson-Crick binding of the antisense oligonucleotide to complementary sequences of the targeted mRNA in cancer cells is able to decrease the gene expression. As a consequence, the introduced antisense oligonucleotide can change the phenotypes of cancer cells and exert anticancer activity via various mechanisms, such as cell cycle arrest, inhibition of anti-apoptosis, inhibition of angiogenesis and potentiation of chemotherapy.

The simplest form of antisense is a small segment of DNA, i.e. a chemically synthetic oligodeoxyribonucleotide (ODN). Several ODNs have been tested in colon cancer treatment in preclinical experiments with or without cytotoxic drug treatment, for example antisense MDM2 combined with cisplatin or topotecan, antisense thymidylate synthase, antisense matrilysin, etc76–78. A clinical trial using an antisense Bcl-2, G3139, in combination with irinotecan in patients with metastatic or recurrent colorectal cancers expressing Bcl-2 was completed last year79. The result has not yet been published.

Transcriptionally active drugs

Inefficient gene transfer and low levels of gene expression often limit the gene therapy strategies described above. Chromatin remodelling is important for the active transcription of expressed genes80. Drugs which modulate this process, such as retinoic acid and histone deacetylase inhibitors can enhance adenoviral transgene expression up to 7-fold in vitro and so may be useful in the clinical development of gene therapy81.

Combinations of adenovirus vectors with colorectal cancer treatments such as chemotherapy and radiotherapy may also modulate transgene expression.

Bacterial vectors for gene therapy

A genetically modified Salmonella typhimurium with two gene deletions altering the bacterial lipopolysaccharide to attenuate its pathogenicity can colonise, replicate in, and lyse tumour cells without toxicity in mice after direct intratumoural injection or even intravenous administration.

A phase I clinical trial with this bacterium (VNP20009) delivered by intravenous injection has demonstrated it to be safe and well-tolerated.

Given that the bacteria accumulate in tumours to very high levels compared to normal tissue, a further modification has been made with the insertion of the CD gene so that it acts as a prodrug activating gene therapy vector. Preclinical studies have verified that this vector given in combination with 5-FC pro-drug is safe and effective. A phase I trial is underway82.


   Challenges
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 Abstract
 Introduction
 Gene therapy trials for...
 Challenges
Conclusions and key points...
 References
 
Current gene therapy strategies need refining to achieve clinically significant efficacy. There are several ways to enhance this novel approach.

Improving the low efficiency of gene transfer

The recent development of replication-competent vectors for cancer gene therapy may improve gene transfer efficiency. These viruses can replicate in tumour cells and cause oncolysis, but also continue to infect surrounding unaffected tumour cells and enhance gene spread. In vivo studies showed increased antitumour efficacy when an oncolytic adenovirus was combined with the HSV-thymidine kinase/ganciclovir enzyme prodrug system and a clinical trial using this approach is under way51.

Improving the potency of antitumour effect

One of the approaches to improve the potency of antitumour efficacy is to combine gene therapy with conventional chemotherapy. An augmented antitumour efficacy has been shown in a combination of ONYX-015 with 5-FU and cisplatin in an animal model with colon cancer and in patients with head and neck tumours in clinical trials57. A synergistic antitumour effect has been reported between the HSV-thymidine kinase/ganciclovir system and a topoisomerase I inhibitor, topotecan, or when oncolytic adenovirus containing suicide gene systems were used in conjunction with radiotherapy68. Another way to augment the antitumour effect is to enhance the by-stander killing effect. These include measures to increase cell–cell communications, to induce anti-angiogenic mechanisms, or to provoke T-cell-mediated immunity to tumour cells83.

Specific gene expression at tumour sites to avoid toxicity to normal tissues

To limit specific gene expression to the site of tumours, tumour- or tissue-specific promoters such as CEA, prostate specific antigen, {alpha}-fetoprotein can be used84. Specific antitumour effects can also based on biological differences in enzymatic activity, such as ribonucleotide reductase, between normal hepatocytes and hepatic metastases85.

Systemic targeting of gene transfer at multiple tumour sites

Viral vectors may be re-targeted to specific tumour receptors to allow systemic targeting of gene transfer to multiple tumour sites. This is currently limited by rapid sequestration by the reticulo-endothelial system before vectors reach their targets.

A further problem is that many vectors can be immunogenic. This may prevent the vectors from repeated administration.

Provoking a strong local inflammatory immune response to tumour site(s) but avoiding an autoimmune reaction to normal cells

A potential pitfall of the immune stimulation strategy is that breaking immune tolerance to self-antigens such as CEA may provoke an autoimmune reaction. The future development of immunogene therapy will not only focus on the enhancement of immune response to tumour cells but also on the restriction of the response within the tumour.

Safety of viral vectors: avoiding overwhelming virus infection

Safety is still a concern, especially when viral vectors are used. For herpes simplex virus, the infection of this virus can be eradicated by antiviral drugs (acyclovir or ganciclovir). However, for adenovirus, viral clearance is dependent on the immune system. Therefore, screening should rule out immunocompromised patients from adenovirus-mediated gene transfer protocols. Safety can be potentially improved by the development of gutless virus86, chimaeric virus87, minivirus88 or complementary oncolytic virus89.

Non-invasive monitoring of transgene expression

It is critical that non-invasive imaging systems are developed that can detect transgene expression in vivo and evaluate the pharmacokinetics of genetic medicines to allow thorough pharmacological studies. Through such analysis, gene therapy efficacy may be better quantified and evaluated90.


   Conclusions and key points for clinical practice
Top
 Abstract
 Introduction
 Gene therapy trials for...
 Challenges
 Conclusions and key points...
References
 
Although the preclinical results of gene therapy for colorectal cancer have been promising, gene therapy is still in its infant stage but it is full of potential. To fulfil its promise, there are possible ways to improve results in the short-term.

The quickest way to establish its position in clinical practice is in combination with existing treatment modalities. The envisaged future practice for cancer treatment may involve a multimodality approach, integrating curative or debulking resection, followed by adjuvant therapies including concurrent or sequential gene therapy, chemotherapy and radiotherapy.


   Footnotes
 
Correspondence to: Dr Daniel Palmer, CRUK Institute for Cancer Studies, The Medical School, University of Birmingham, Vincent Drive, Edgbaston, Birmingham B15 2TF, UK


   References
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 Abstract
 Introduction
 Gene therapy trials for...
 Challenges
 Conclusions and key points...
 References
 

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