GENE THERAPY, RECENT DEVELOPMENT AND FUTURE PROSPECTS IN GASTROINTESTINAL ONCOLOGY: A REVIEW ARTICLE

GENE THERAPY, RECENT DEVELOPMENT AND FUTURE PROSPECTS IN GASTROINTESTINAL ONCOLOGY: A REVIEW ARTICLE

Kotla Ramya SreeandMudavath Hanumanaik

Department of Pharmaceutical Chemistry KLE University College of Pharmacy, Vidyanagar, Hubli, Karnataka, India

ABSTRACT

The introduction of nucleic acids into cells has as a purpose of medical condition or disease. Currently, gene therapy studies a broad range of potential therapeutic interventions, including the body's immune reaction to tumours, new blood vessels in the heart to alleviate heart attacks and to stop HIV-replication in patients with AIDS¹ .There is also renewed emphasis on the gene therapy of genetic diseases, such as haemophilia A and B, and cystic fibrosis. Human gene therapy experimentation raises many issues. In this review article, background of gene therapy, introduction, genetic diseases, gene function, germ line gene therapy, hurdles in gene therapy, methods for gene therapy, ex vivo, in vitro and in vivo-gene therapy, risks associated with gene therapy, have been given.

INTRODUCTION: Advances in the molecular biology have been made early in the 1980. It has been already studied that human genes can be sequenced and cloned. Scientists search new methods for easily producing of proteins, such as insulin in diabetic patients. Modified bacteria, introduced in the body, can be harvested and injected in people, who cannot produce it naturally. Scientists try to introduce genes straight into human cells, focusing on diseases, caused by single-gene defects, such as cystic fibrosis, haemophilia, muscular dystrophy and sickle-cell anaemia ².

Gene therapy for haemophilia B other hereditary plasma protein deficiencies have shown great promise in pre-clinical and early clinical trials ³. Gene therapy can be broadly defined as a transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapies is to transform viruses into genetic shuttles, which would deliver the gene of interest into the target cells.

Based on the nature of the viral genome, these gene therapy vectors could be divided into RNA and/or DNA viral vectors. The majority of RNA virus based vectors have been derived from simple retroviruses like murine leukaemia virus. A major shortcoming of these vectors is that they are not able to transduce non-dividing cells. This problem may be overcome by use of novel retroviral vectors, derived from lentiviruses, such as human immunodeficiency virus (HIV) ⁴.

The most commonly used DNA virus vectors are based on adenoviruses and adeno-associated viruses (AAVs). Although, the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary. Gene transfer technologies are promising tools to manipulate donor T-cell immunity to enforce graft-versus-tumour/graft-versus-infection, while prevention or control of graft versus host disease.

For this purpose, several cell and gene transfer approaches have been investigated at the pre-clinical level and implemented in clinical trials ⁵. The nuclear envelope represents a key barrier to successful non- viral transfection and gene therapy both in vitro and in vivo. Although the main purpose of the nuclear envelope is to partite the cell to maintain cytoplasmic components in the cytoplasm and nuclear components,

The most notably genomic DNA in the nucleus, this function poses a problem for transfections, in which exogenous DNA is delivered into the cytoplasm. After delivery to the cytoplasm, nucleic acids rapidly become more complex, with cellular proteins that mediate interactions with the cell machinery for their traffics. Thus, these proteins are that, in essence, which control the nuclear import of DNA, and we must also understand their activities in cells ⁶.

Gene therapy for neurological, and in particular, neurodegenerative, diseases, is now a reality. A number of early phase clinical trials have been completed and several are currently in progress. In view of this, it is critically important to be evaluated the immunological risk, associated with neurological gene therapy, which has clear implications for trial safety and efficacy. Moreover, it is imperative in particular to identify factors, indicating potential high risk ⁷. Viral vectors are potent gene-delivery platforms, used for the treatment of genetic and acquired diseases.

However, just as viruses have evolved to infect cells efficiently, the immune system has evolved to fight off what it perceives as invading pathogens. Therefore, innate immunity and antigen-specific adaptive immune responses against vector-derived antigens reduce the efficacy and stability of in vivo-gene transfer.

In addition, a number of vectors are derived from parent viruses that humans encounter through natural infection, resulting in pre-existing antibodies and possibly in memory responses against vector antigens. Similarly, antibody and T-cell responses might be directed against therapeutic gene products that often differ of the endogenous non-functional or absent protein that is being replaced.

As details and mechanisms of such immune reactions are uncovered, novel strategies are being developed, and vectors are being specifically engineered to avoid, suppress and/or manipulate the response, ideally resulting in sustained expression and immune tolerance to the transgene product ⁸.

Haematopoietic stem cell transplantation (HSCT) is now widely used for treatment of primary immune deficiencies (PID). For patients with specific disorders (severe combined immunodeficiency (SCID)- X1, adenosine de aminase deficiency (ADA)–SCID, Xchronic granulomatous disease (CGD) and Wiskott–Aldrich Syndrome (WAS), who lack a suitable human leukocyte antigen- (HLA)-matched donor, gene therapy has offered an important alternative treatment option ⁹. Artificial chromosomes (ACs) are highly promising vectors for use in gene therapy applications ¹⁰.

They are able to maintain expression of genomic-sized exogenous transgenes within target cells, without integrating into the host genome. Although, these vectors have huge potential and benefits, in comparison with normal expression constructs, they are highly complex, technically challenging to construct and difficult to deliver to target cells ¹¹. In the last two decades, remarkable advances have been made in the development of technologies used to engineer new aptamers and ribozymes. This has encouraged interest among researchers, who seek to create new types of gene-control systems that could be made to respond specifically to small-molecule signals.

Validation of the fact that RNA-molecules can exhibit the characteristics, needed to serve as precision genetic switches, has come from the discovery of numerous classes of natural ligand sensing RNAs, called ribo-switches. Although a great deal of progress has been made toward engineering useful designer ribo-switches, considerable advances are needed before the performance characteristics of these RNAs match those of protein systems that have been  regulate gene expression ¹².

Pulmonary gene therapy cures diseases such as cystic fibrosis, α1-antitrypsin deficiency, lung cancer and pulmonary hypertension. Efficient expression of delivered genes in target cell types is essential for the achievement of this goal ¹³.

Genetic Diseases: Cystic fibrosis, blood disorders, muscular dystrophy and diabetes.

Understanding Gene Function: From the estimated 30 to 50,000 genes, we know the function of a very few. Attempting gene therapy how every one of them works could address only some of the genes, implicated in particular diseases. Likewise, genes may have more than one function.

Germ Line Gene Therapy: This technique involves the genetic modification of germ cells. Such therapy would change the genetic makeup of the egg or sperm of an individual and would be carried onto future generations. This would offer the possibility of removing an inherited disorder from a family line forever.

Hurdles in Gene Therapy: The therapeutic genes are inserted into the body through specific constructs, called vectors, which deliver therapeutic genes to the patients’ cells. The most common vectors are viruses. Scientists try to manipulate the viral genome to remove the disease-causing genes and introduce therapeutic genes. The introduction of viruses in the body might cause side effects like toxicity, immune and inflammatory responses, as well as gene control and targeting issues.

Principles of Gene Therapy: Selection of Agene, A Vector and A Management strategy:

Selection of the gene: Mutant gene correction. The principles of gene selection strategies are illustrated in Figure 1. In the case of inherited monogenic diseases, the aim of gene therapy is to transfer and express the defective gene. The situation is more complex in cancer gene therapy because cancer most often results from sequential genetic and epigenetic alterations, affecting oncogenes, tumour-suppressor genes and micro RNAs.

One gene therapy approach is, thus, to restore tumour-suppressor gene expression or to inhibit oncogene expression. About 11% of transferred genes in gene therapy clinical trials are tumour-suppressor genes and many trials have been performed in cancer gene therapy using the p53 gene, mostly including patients with lung or head and neck cancers.

Suicide Genes: The aim of suicide gene therapy is to enable, selectively, the transfected cell to transform a Prodrug into a toxic metabolite, resulting in cell death. The most widely described suicide gene is the herpes simplex virus thymidine kinase (HSV-tk) gene. HSV-tk can phosphorylate ganciclovir, which is poor substrate for mammalian thymidine kinase. Ganciclovir can therefore be transformed into ganciclovir triphosphate, which is cytotoxic to the transfected cell; resulting in celldeath ¹⁴. This cell death can also affect neighbouring cells that do not express HSV-tk. This phenomenon is called a local bystander effect; as opposed to a bystander effect that can be observed in distant, nontransduced tumour sites ¹⁵. This distant bystander effect involves the immune system.

Immunotherapy: Cancer immunotherapy has been developed to stimulate immune response against tumour cells. Gene therapy can be used to transfer genes into tumour cells to render them more highly immunogenic. Gene transfer of tumour-specific antigens, co-stimulatory molecules and⁄or inflammatory cytokines has been assessed. Tumour-associated antigens can be recognized by T lymphocytes. These antigens can be derived from oncogenic viruses (Epstein–Barr virus, human papilloma virus) or can be self-antigens. These self-antigens can be over expressed antigens or antigens that are altered by virtue of a gene mutation or a post-translational modification. They can also be onco-foetal antigens, such as5T4 antigen and carcino embryonic antigen (CEA) ¹⁶. Immunization against a specific antigen can induce cellular and ⁄ or humoral immune responses.

T-cell activation requires not only the interaction between major histocompatibility molecules bearing a specific peptide and the T-cell receptor, but also non-antigen-specific co-stimulatory activation by interaction of molecules expressed on the T-cell and the antigen presenting cells (such as interactions between CD28 and CTLA-4 expressed on the T-cell and B7 expressed on the antigen presenting cells). Finally, vectors encoding inflammatory molecule genes [such as interleukin-2, interleukin-12, TNF-a, interferon-c, granulocyte macrophage colony-stimulating factor (GM-CSF)] have been engineered. In terms of the site of gene transfer, immunization can be carried out in situ in the tumour or at distant site ¹⁷.

RNA interference: RNA interference is a promising new therapeutic approach for many diseases, including cancer. Micro RNAs (miRNA) are short, endogenous _22 nucleotide RNAs. They can regulate gene expression at the post transcriptional level by binding to the 3’-Un translated region of the target mRNA, resulting in either mRNA degradation or inhibition of translation ^{18, 19}. They act as fine-regulators of the proteome ²⁰. miRNAs can have oncogenic activities when they are up regulated and target tumour suppressor genes ²¹. miRNAs can also have a tumour suppressor potential ²². Furthermore, miRNA scan modify the response to therapeutic agents. For example, the Let-7 family of miRNAs can modify the response of cancer cells to radiation therapy²³. The endogenous RNA interference pathway has been exploited to develop other RNA interference molecules: synthetic, exogenous, double stranded, short, interfering RNA (siRNA) and vectors expressing short hairpin RNAs (shRNA). As opposed to siRNA, shRNA are synthesized in the nucleus and use maturation pathways similar to the miRNA maturation pathways ²⁴. As cancer cells present many well-known RNA interference targets, there are multiple opportunities for therapeutic gene silencing in oncology ²⁵.

FIGURE 1: PRINCIPLES OF GENE THERAPY. (a) Gene re-expression. The vector carries a wild-type version of a mutant gene into the tumour. (b) Suicide therapy. A combination of the systemic administration of a nontoxic prodrug and the tumour-specific delivery of the prodrug-activating enzyme gene. (c) Immune therapy. The vector carries a gene encoding specific immunogenic tumour antigen (1), a co-stimulatory signalling molecule (2) or an inflammatory cytokine (3), leading to immune stimulation. (d) Oncolytic viruses. Oncolytic viruses specifically replicate in, and kill, tumour cells. (e) Therapeutic RNA interference. The RNAi (siRNA) or the RNAi gene (sh RNA) is delivered into the cell. After maturation and incorporation in the RNA-induced silencing complex (RISC), RNAi can bind an oncogene RNA, leading to oncogene repression and cell death.

The choice of the Vector: The effectiveness of gene therapy is highly dependent on the efficacy of gene transfer. The vector has to be safe, has to protect the genetic material from degradation in the extracellular environment, and must release the genetic material to the target cell.

Viral Vectors: Recombinant viruses have been shown to be efficient for gene transfer both in vitro and in vivo. Many viruses such as adenoviruses, adeno-associated viruses (AAVs), retroviruses, lentiviruses, herpes viruses, poxviruses, measles viruses, simian virus 40 recombinant (SV40r) and vesicular stomatitis viruses have been proposed for preclinical studies (Table 1). Other viruses’ are used as virotherapy, rather than gene therapy agents, because they cannot be genetically modified (reoviruses) or because they have not been modified with the insertion of a therapeutic transgene. Data and clinical trials related to these viruses are not reported in this review. Viruses can be either replication-defective or replication-competent. To engineer replication-defective viruses, one or several genes required for viral replication can be deleted. Vectors that are able to replicate selectively in cancer cells would be of great interest in oncology.

A new strategy involving so-called ‘Oncolytic’ viruses has been proposed. In this context, the aim is to generate a pathogen for the tumour, which is unable to propagate in, and to harm, normal tissues. Two main characteristics of Oncolytic viruses are (i) they replicate selectively in cancer cells and have self-amplification properties; and (ii) they have cancer-cell-specific toxicity ²⁶.

Some viruses, such as reoviruses, have natural, inherent tumour selectivity ^{27, 28}. From a general point of view, Oncolytic viruses can be engineered in three ways. (i) Viruses can be deleted for genes that are necessary for their replication in normal cells, but not in cancer cells. (ii) Another possibility is to engineer a virus with genes under the control of a tumour-specific promoter. (iii) Viruses can also be engineered to bind to specific tumour antigens on the cell surface ²⁹. In the first two approaches, the aim is to restrict the replication of the viruses, whereas the third strategy aims at selective infection of cancer cells. However, viral vectors have many limitations. There is a limiting immunogenicity and there are safety considerations, including a possible intentional mutagenesis for retroviruses and lentiviruses.

TABLE 1: SOME ADVANTAGES AND DISADVANTAGES OF DIFFERENT GENE THERAPY VECTORS

Non viral vectors:

1. Naked DNA: Naked or plasmid DNA has a rapid clearance and a low cellular uptake. As a consequence, the main application of naked DNA injection is in vaccination and immunotherapy strategies. Electroporation involves the generation of a transient increase of the permeability of cell membranes using electric pulses and is more efficient than a simple injection of naked DNA. Electroporation increases the transfer efficiency of drugs, DNA or RNA into the cells, in vitro and in vivo in the case of intra tumoural injections ^30-32. However, the safety and efficiency of this procedure need to be assessed in humans.
2. Synthetic vectors: Cationic liposomes comprise a lipid bilayer membrane. They are amphiphilic molecules and can interact with negatively charged DNA, hereby making it compact and protecting it from circulating endonucleases. The structure generated upon incubation of DNA with liposome is called a lipoplex. Cationic lipoplexes can bind to negatively charged cell membranes and induce cellular uptake. However, in vivo transfer efficiency remains low ³³. Blood vessels in tumours can have gaps between endothelial cells, up to 800 nm in size.

Nanoparticles can extravagate through these gaps and accumulate selectively in tumours. This enhanced permeability and retention (EPR) effect could explain the tumour selectivity of several gene therapy vectors ³⁴. Nanoparticles are small-sized vectors that can be lipoplexes, other polymers, or metal- and ceramic-based. They can be used as gene therapy vectors and can accumulate in tumours as a result of the EPR effect ^{35, 36}.

3. Biological non viral vectors: Bacteria can be used as gene therapy vectors. They can be used as gene transfer vectors or protein delivery systems when the therapeutic transgene is already expressed in the bacterium ³⁷. In addition, many mammalian cell types can be used as carriers of gene therapy vectors ³⁸.

Haematological and progenitormesenchymal cells are promising agents for use in cancer gene therapy. For example, CIK (cytokine induced killer) cells can be obtained from mousses plenocytes or human peripheral blood, after in-vitro stimulation and culture with interferon-c, interleukin-2and anti-CD3 antibodies. These cells naturally target tumours and have cytotoxic effects. They have been combined with the Oncolytic vaccinia virus and a synergistic antitumor effect has been observed in mouse models ³⁹.

4. Choice of strategy: In vivo vs. ex vivo. The ex vivo strategy consists of collecting a cell type or its precursor from the subject, culturing and transducing the cells with the vector and then reintroducing the genetically modified cells into the subject. The in vivo strategy consists of direct injection of the vector into the subject (either intra tumourally or systemically). The ex vivo strategy allows control of the transduction and limitation of dissemination of the vector. The in vivo strategy does not require a cell culture step, but cell targeting and transduction efficiency are more difficult to control.

Gene Therapy in Gastrointestinaloncology:

Clinical Trials and Future Prospects:

Liver tumours:

Clinical trials: Based on preclinical data, several clinical phase I and phase II trials concerning gene therapy of liver tumours have been published (Table 2) ^40-47. Re-expression of tumour-suppressor genes has been evaluated in vitro, in vivo and in clinical trials, mainly with vectors encoding wild-type p53. In a recently published, phase II trial, 46 patients with unrespectable hepato cellularcarcinoma were randomized to either group 1 (multiple hepatic arterial injections of 5-FU and a recombinant adenovirus encoding the p53 gene, after transcatheterarterial chemoembolization (TACE)) or group 2 (TACE alone). Both treatments were safe. Partial response or stable disease was reported for 69.5% of subjects in group 1 and 65.2% of subjects in group 2. Times to progression were 9.6 months in group 1 and 8.3 months in group 2.

Overall survival times were 12.8 months in group 1 and 10.4 months in group 2 ⁴⁷. Other gene therapy trials have considered suicide therapy strategies. A recent, phase II clinical trial compared patients with hepatocellular carcinoma and tumours bigger than 5 cm, treated by liver transplantation, with or without adenoviral-mediated HSV-tk therapy. During trans-plantation surgery, adenoviruses encoding HSV-tk were injected into the peritoneal cavity around the liver and ganciclovir therapy was started 24 h after surgery and continued for 10 days afterwards. Forty-five patients were included in the trial, 22 received liver transplantation only and 23 received liver transplantation and gene therapy. Recurrence-free survival and overall survival were significantly higher in the gene therapy group, particularly in patients without vascular invasion before treatment ⁴⁵.

A phase I trial included 21 patients (nine patients’ with primary liver cancers, five with metastatic colorectal cancers and seven with pancreatic cancer) in an immunotherapy approach. Intratumoural injections of an adenovirus encoding the interleukin 12 gene were well tolerated, and a partial, objective remission was observed in one patient with hepatocellular carcinoma ⁴³. Another phase I trial included 17 patients (nine patients with primary liver cancers, five with colorectal cancers and three with pancreatic cancer). Patients were treated with Intratumoural injections of autologous dendritic cells transfected with an adenovirus-encoding IL-12 gene. The treatment was well tolerated. It was associated with a marked increase of infiltrating CD8+ T lymphocytes in three of 11 tumour biopsies analysed.

TABLE 2: CLINICAL TRIALS CONCERNING GENE THERAPY IN PRIMARY LIVER TUMOURS

PR, partial response; SD, stable disease; PD, progressive disease; RFS, recurrence free survival; OS, overall survival.

Eleven patients could be assessed for response. One partial response was observed in a patient with pancreatic carcinoma. Stable disease was observed in two patients and progression in eight patients ⁴⁴. Oncolytic viruses have also been evaluated in clinical trials. In a phase II study, 19 patients with advanced or metastatic hepatobiliary cancers (tumours of the liver, gall bladder, or bile duct) received Intratumoural injections of ONYX-015, a first-generation, replication selective adenovirus. Sixteen patients could be evaluated for response. One patient had a partial response, one patient had prolonged disease stabilization (49 weeks), and eight patients had a biological response with a decrease in tumour markers of more than 50% ⁴¹.

Recent clinical studies with other therapeutic viruses have been reported, highlighting the interest in Oncolytic viruses for cancer gene therapy. A phase I trial, including 14patients with refractory primary or metastatic liver tumours, evaluated the maximum tolerated dose of Intratumoural injection of JX-594. JX-594 is a targeted, Oncolytic poxvirus (vaccinia virus) modified by disruption of the thymidine kinase gene and insertion of the human GM-CSF.

Patients received a mean of 3.4 cycles of Intratumoural injections. Four patients received only one cycle (two because of toxicity and two because of self withdrawal from the study) and were not analysed for tumour response. Among the 10 patients analysed, three showed a partial response, six had stable disease and one showed progression of disease after CT-scan examination. Hyperbilirubinaemia was a limiting factor in patients treated with the highest dose. Flu-like symptoms were the most frequent adverse event. Of particular interest, JX-594 was found in peripheral blood after the initial injection and delayed viraemia was detected several days afterwards. JX-594 was found in non-injected tumours, suggesting that the virus could target disseminated tumours after systemic injection ⁴⁶. Another study included three patients with concomitant hepato cellularcarcinoma and hepatitis B virus infection (HBV) with active viral replication. JX-594 intratumoural injections were associated with tumour response and a decrease in HBV DNA levels ⁴⁸.

Perspectives from Preclinical Models: New strategies aiming at improving the rate of transduction of cells have been developed for gene re-expression strategies, with interesting preclinical results. For example, an adenoviral vector encoding a fusion protein, p53-VP22, has been engineered. VP22 is a HSV type 1 tegument protein, exhibiting the capacity to be exported from the cell to neighbouring cells. Some VP22 fusion proteins, such as p53, can retain this capacity. In a model of heterotrophic, subcutaneous hepatocellular carcinoma xenograftin mice, intratumoural injections of VP22-p53 adenoviruses were more efficient than those of p53-aloneadenoviruses, leading to partial tumour regression ⁴⁹.

The most widely described suicide gene is the HSV-tk gene. However, other suicide gene strategies have been developed. For example, an adenoviral vector encoding carboxypeptidase G2 (CPG2) has been engineered. CPG2is an enzyme that catalyses the conversion of alkylating prodrug such as ZD2767P into cytotoxic agents. Tumour regression was observed in a model of subcutaneous hepatocellular carcinoma xenograft, after Intratumoural injection of the vector and treatment with the prodrug, ZD2767P ⁵⁰. Other immune therapy strategies have also been evaluated in vitro.

Adenoviruses encoding CD40 ligands were injected into orthotropic hepatocellular carcinomas in rats. The treatment caused a tumour response, with 10 of 23 treated animals showing a complete response and nine animals a partial response, and an improved survival of treated rats. The treatment induced lymphocytic infiltration of the tumour, and the tumour response was dependent on CD8+ T cells ⁵¹. In a model of alpha-fetoprotein-expressing hepatocellular carcinoma spontaneously developing in mice (BW7756 tumours), the combination of alpha-fetoprotein vaccination (by injection of expression plasmids into muscles) and an adenoviral-mediated chemokine IP-10 and interleukin-12 expression (intratumoural injection) resulted in a synergistic effect with improved survival ⁵².

The sodium-iodide symporter (NIS) gene encodes a membrane glycoprotein that mediates active iodide transport across the basolateral membrane of thyroid follicular cells. NIS can drive the uptake of radioisotopes (123Iodide, 124Iodide, 125Iodide, 131Iodide, 188Rhenium,

211Astatine) into the cells ⁵³. A viral-guided NIS gene therapy is an innovative approach of radio-iodine therapy. Such an approach was assessed in a rat model of hepatocarcinoma (diethyl nitrosamine-treated rats). Animals were treated by intraportal injections of an adenovirus encoding rat NIS. Long-term retention of radioiodine was demonstrated after viral transduction, both in treated, tumour-bearing rats and in healthy, treated rats, but not in control, non treated rats. After Intratumoural injection of the virus, intraperitoneal injections of 131I were associated with inhibition of tumour growth and prevention of the formation of new tumour nodules around the injected nodule. Furthermore, intraperitoneal injection of the virus followed by 131I therapy was associated with improved survival of diethylnitrosamine-treatedrats⁵⁴.

New strategies include antiangiogenic gene therapy and therapeutic RNAi. Antiangiogenic therapies are currently well developed and widely used in oncology. Endostatinis an inhibitor of angiogenesis and tumourgrowth ⁵⁵. An adenovirus encoding human endostatin was tested in a model of subcutaneous hepato cellularcarcinoma xenograft in mice and demonstrated an antitumoureffect ⁵⁶. Pigment epithelium-derived factor (PEDF) is another recently discovered, antiangiogenic factor. In a model of heterotopic, subcutaneous hepato cellularcarcinoma xenograft in mice, intratumoural injections of a plasmid expressing PEDF resulted in a significant reduction of tumour volume compared withcontrols ⁴⁴. Kallistatin is another example of new antiangiogenic inhibitors. Intraportal injections of a recombinant AAV encoding kallistatin were performed in hepatic and subcutaneous hepatocarcinoma murine models. Treatment with the recombinant virus resulted in significant tumour growth inhibition, as compared with treatment with empty AAV ⁵⁸.

There has been recent interest in the RNAi strategy. The pituitary tumour-transforming gene 1 (PTTG1) is an oncogene, which is frequently expressed in human liver cancers. An adenovirus encoding an RNAi targeting PTTG1 (Ad.PTTG1-siRNA) has been engineered. In vitro, the virus could deplete PTTG1 in hepatoma cells, resulting in apoptosis. Furthermore, intratumoural injections of the virus led to inhibition of tumour growth in a xenograft tumour in mice ⁵⁹. Another recent study concerns RNAi cancer gene therapy in a mouse model of

hepatocellular carcinoma (test-O-MYC; LAP-tTa). Hepato cellularcarcinoma cells exhibit a down regulation of the expression of miR-26a in human and mouse liver tumours. This miRNA plays a role in cell cycle arrest. Systemic administration by tail vein injection of an adeno-associated virus encoding miR-26a resulted in the inhibition of cancer proliferation, with six of eight treated mice exhibiting only small tumours or no tumour and six of eight mice treated with control virus developing a fulminate disease⁶⁰.

Colorectal cancer:

Clinical trials. Clinical trials concerning the gene therapy of metastatic colorectal cancers have also been published (Table 3) ^61-68. Re-expression of tumour suppress or genes has been evaluated in a phase I trial including 17 patients with advanced cancers, among them five patients with colorectal cancer and one patient with pancreatic cancer. Patients were treated with escalating doses of an adenovirus encoding a wild-type p53gene (Ad5CMV-p53). The treatment was administered intravenously, daily for 3 consecutive days, every 28 days.

There were no objective responses; one patient with metastatic and progressive colorectal carcinoma had a 10-month period of stable disease. The virus was detectable in plasma 14 and 28 days after treatment in the majority of patients treated at the highest dose. Seven patients had tumour biopsies before and after treatment. Six of these patients had undetectable Ad5CMV-p53expression before treatment that became detectable after

A phase I clinical trial including 16 patients with hepatic metastases from colorectal carcinomas assessed the safety of intratumoural injections of an adenovirus encoding the HSV-tk gene. Injections were followed by systemic ganciclovir administration. The treatment was safe. However, there was no complete or partial tumourresponse ⁶². P53-specific immunity can be observed in advanced cancers. In a phase I⁄II, dose-escalation trial, 15 patients with advanced colorectal cancers were treated with systemic injections of a poxvirus encoding a wild-type p53gene, aiming to induce p53-specific immunity. The treatment could induce p53-specific antibodies and, in two of five patients treated with the highest dose of virus, a specific T-cell response ⁶⁴. TROVAX is a modified vaccinia virus encoding the tumour antigen, 5T4, aiming to produce an anti-5T4 immune response. Trovax is used in several cancer gene therapy protocols. A phase I ⁄ II trial was carried out in 22 patients with metastatic colorectal cancer. Repeated intramuscular injections of TROVAX were safe and induced an anti-5T4 cellular response. Five of 22 patients had stable disease for a period ranging from 3 to 18 months ⁶⁷. A 5T4-specific cellular or antibody response was still detectable when given alongside a chemotherapy regimen ⁶⁹.

A phase II trial included 27 patients with colorectal cancer liver metastases. Infusions of ONYX-015 into the hepatic artery, in combination with 5-FU, were well tolerated with fever, rigours and fatigue as the most common side effects. The antitumoural effect of the virus could not be assessed in patients who had not previously received this chemotherapy regimen. Interestingly, three patients with 5-FU-refractory disease had a 30–50% regression of the tumour mass⁶⁵. Another phase I, dose-escalation trial included 12 patients with liver colorectal cancer metastases. All patients were refractory to a first line chemotherapy. NV1020 is an oncolytic HSV type 1. Patients had a single NV1020 injection into the hepatic artery at four different doses (three patients in each group).

TABLE 3: CLINICAL TRIALS CONCERNING GENE THERAPY IN METASTATIC COLORECTAL TUMOURS

PR, partial response; SD, stable disease; PD,progressive disease.

NV1020 administration was well tolerated with fever, rigours and headache as the most common side effects. Antitumor activity was assessed by CT-scan at day 28, even if this was a toxicity study. Two patients had a 39% and a 20%reduction respectively, in tumour size; seven patients showed stable disease and three progressive disease ⁶⁸. Floxuridin chemotherapy was administered after day 28 by an intra hepatic arterial pump, in association with intravenous irinotecan. All 12 patients had a partial response to subsequent chemotherapy ⁷⁰.

Perspectives from preclinical models. In a recent study, ap53 gene re-expression strategy was improved in a combination of viral-directed therapies. An AAV vector expressing the cringle 1 domain of the human hepatocyte growth factor (AAV-HGFK1) and an adenovirus expressing p53 were combined in a murine model of colorectal cancer xenograft. The combination treatment increased the survival time, inducing apoptosis, necrosis and suppressing angiogenesis, as compared with single agent therapies.

In vitro, this antiangiogenic effect was related to an inhibitory effect on endothelial cell migration ⁷¹. The HSV-tk⁄ ganciclovir suicide strategy could also be improved. An adenovirus encoding the monocytechemoattractant protein-1 (MCP-1) gene has been engineered. In a model of subcutaneous colorectal cell line tumours in immune competent mice, intratumoural injections of both MCP-1-encoding adenovirus and of HSV-tk-encoding adenovirus reduced tumour growth significantly more than HSV-tk adenovirus alone ⁷². Another suicide system has been developed in a murine model of colorectal cancer. A retrovirus encoding the cytosine deaminase (CD) gene was engineered. CD converts the nontoxic 5-fluorocytosine into toxic 5-fluorouracil. In a model of orthotropic liver metastasis (CT26cells) in mice; the viral vector was administered into the portal vein via intrasplenic injection.

5fluorocytosinewas administered into the peritoneal cavity. All mice in the control groups had tumour progression, but tumour growth was significantly inhibited in the treated mice ⁷³. Chemo-gene and radiation-gene therapies are the combinations of gene therapy with chemotherapy or radiation therapy respectively. Gene therapy can sensitize colon cancer cells to chemotherapy in vitro ^{74, 75}.

CD converts 5-fluorocytosine into the toxic metabolite, 5-fluorouracil. Intratumoural injection of free plasmids expressing CD in liver metastasis rat models, in association with an oral treatment with 5-fluorocytosine, resulted in a systemic antitumor effect ⁷⁶. Flt3 Ligand (Flt3L) is an activator of dendritic cells and NK cells. Intratumoural injections of an adenovirus encoding Flt3Lproduced a synergistic effect in association with 5-FUtherapy in subcutaneous models of hepatocarcinoma and colon cancer and induced long-term immunity against further parental cancer cell injections ⁷⁷. In a recent study, cultured human colorectal cancer cells and tumour xenograft were infected with an engineered adenovirus expressing the NIS gene. Tumour xenografts concentrated 99m Technetium, allowing quantitative imaging by SPECT ⁄ CT. NIS expression reached a peak 48 h after intratumoural injection. Administration of a single dose of virus combined with a single 131I dose 48 h after virus injection induced tumour regression with an additionaleffect ⁷⁸.

Recently, the effect of miRNA targeting c-myc was assessed in vitro and in vivo. In vitro, miRNA inhibitedc-myc expression reduced HT 29 cell proliferation and induced apoptosis. In vivo, intratumoural injections of miRNA were performed in a model of subcutaneous HT  29 tumour in athymic nude mice. Treatment significantly reduced tumour volume ⁷⁹. In another study, a replication-deficient adenovirus expressing p53 and a miRNA targeting the cyclin-dependent kinase inhibitor p 21(Adp53 ⁄ mIR-p21) have been engineered. In tumour xenograftsin nude mice, intratumoural injections resulted in a significant decrease in tumour volume, as compared with a control virus expressing p53, but not mIR-p21.

In vitro, Adp53 ⁄ mIR-p21 increased apoptosis as compared with the control virus, but also increased the sensitivity of cancer cells to doxorubicin ⁸⁰.

Pancreatic cancer:

Clinical trials: Clinical studies have also been carried out in pancreatic cancer (Table 4) ^68-71. A phase I study assessed the safety and side effects of a subcutaneous vaccination regimen using two viruses (vaccinia virus and fowl pox virus) expressing CEA and mucin-1 with three co-stimulatory molecules (B7.1, ICAM-1 and LFA-3), in association with GM-CSF therapy, in 10 patients with advanced pancreatic cancer. The most common side effects were injection site reactions, fatigue, headache and nausea. Survival was significantly better in patients who developed anti-CEA- or anti-MUC-1-specificimmune responses; however, these are preliminarydata ⁷⁰. A phase III study in metastatic pancreatic cancer has been carried out (US 635 phase III trial), but results have not yet been published.

A phase I⁄II study showed the feasibility and tolerability of endoscopic ultrasound-guided, repeated administrations of the ONYX-015 virus into carcinomas of thepancreas ⁸². An interim analysis of the PACT study, a multicentre, randomized, controlled, phase III clinical trial, was presented at the American Society of Clinical Oncology meeting 2009. The study included 330 patients with advanced pancreatic cancers, with two arms of treatment.

The control arm received standard, 5-week chemo radiation therapy, the experimental arm received chemo-radiation plus weekly, intratumoural injections of TNF-erade (Genvec, Inc., Gaithersburg, Maryland, USA), an adenovirus encoding the TNF-a gene. Both arms received adjuvant gemcitabine with the option of erlotinib. An interim analysis of overall survival was conducted after the 92^nd death (one-third of total expected events) had occurred. The analysis concluded that TNF-erade appeared to be safe and well-tolerated. The overall survival interim analysis indicated a trend in favour of TNF-erade therapy, with a late effect on Kaplan Meyer curves ⁸⁵.

TABLE 4: CLINICAL TRIALS CONCERNING GENE THERAPY IN PANCREATIC CANCER

PR, partial response; SD, stable disease.

Perspectives from preclinical models: Transduction efficiency is a major concern in gene therapy. A recently published study showed high transduction efficiency for gene transfer in cells derived from pancreatic cancers, using a lentiviral vector, both in vitro (from 68% to 98%, depending on the cell line and the multiplicity of infection) and in vivo. In tumour xenografts in mice, intratumoural injections of a lentivirus encoding human interferon-b resulted in a significant inhibition of apoptosis ⁸⁶.

Peritoneal dissemination is a poor prognostic event in pancreatic cancer. Intraperitoneal vector injections may therefore be of great interest. Intraperitoneal injection of retrovirus encoding the HSV-tk gene has been effective in treating nude mice with a model of metastatic pancreatic cancer disseminated to the peritoneal cavity ⁸⁷. The K-rats oncogene is mutated in more than 80% of ductal pancreatic adenocarcinomas ⁸⁸. An adenovirus expressing K-rats antisense RNA can induce apoptosis in vitro in human pancreatic cancer cells. Intraperitoneal injections of this adenovirus inhibited peritoneal dissemination in hamster pancreatic cancer models ⁸⁹.

We will focus on the promising approach of chemotherapy and radiation gene therapy. Adenoviruses encoding p53 genes showed a synergistic antitumour effect with 5-FU therapy, in vitro and in vivo, in heterotopic models in rats ⁹⁰. A recombinant adenovirus encoding deoxycytidine kinase and uridine monophosphate kinase (enzymes converting gemcitabine into toxic metabolites) sensitized pancreatic cancer cells to gemcitabine, in vitro and in an orthotopic tumour hamster model ⁹¹.

In a subcutaneous model of pancreatic carcinoma in mice, a single, intravenous injection of the vaccinia GLV-1h68 virus, an oncolytic, engineered poxvirus, was able to reverse the course of tumour growth. The efficacy of viral therapy was enhanced by repeated injections of gemcitabine or cisplatin. Seven of eight mice treated with the virus and cisplatin had complete tumour regression at the end of the observation period, whereas one of eight mice treated with the virus alone had complete tumour regression. The viral therapy was safe, with a high tumour selectivity of the virus after systemic administration ⁹².

Another study demonstrated that an Oncolytic virus was able to synergize with gemcitabine to kill pancreatic cancer cells in vitro and in vivo by a synergistic effect on apoptotic pathways ⁹³. In another study, cultured human pancreatic cancer cells and tumour xenografts were infected with an engineered measles virus expressing the NIS gene. Intratumoural injections resulted in a significant reduction in tumour volume and increased survival time of the treated mice compared with the control mice. Tumour xenografts also concentrated radioiodine, allowing quantitative imaging with 123I micro-SPECT⁄ CT ⁹⁴. Pancreatic cancer is therefore a candidate for specific, viral-driven delivery of therapeutic radioisotopes.

CONCLUSION: The safety profile of gene therapy is a major concern. Recently published trials have shown good safety records. The most commonly reported side effects related to treatments involving a viral vector were fever, headache, fatigue, nausea and vomiting. Intratumoural injections were used in most studies.

In trials using replication competent agents, viruses were found in non-injected tumours and regression of both injected and distant disease occurred. This effect could be related to targeting of disseminated tumours by the vector and to local and systemic immune responses.

Thus, intratumoural injections could be efficient, even for the treatment of disseminated tumours. However, lesions can be difficult, or impossible, to inject, and repeated intratumoural injections using invasive procedures could be a limiting factor for this approach. Systemic administration has also shown a good safety record and the development of oncolytic viruses that selectively target tumour cells makes this mode of administration of great interest. These data regarding oncolytic viruses may pave the way for more widespread clinical applications of this approach. Gene therapy will need to be positioned in the context of existing cancer therapy strategies.

Good safety records could make gene therapy a good candidate in all therapeutic settings (neo-adjuvant, adjuvant and advanced disease), probably in combination therapies. Combining gene therapy with chemotherapy or radiation therapy may be very interesting, as gene therapy can act as a chemo- sensitizer or radio-sensitizer, and because chemotherapy and radiation therapy can improve gene transfer efficiency and gene expression ^95-99. Gene therapy is not yet suitable for use in routine clinical practice, mainly because of insufficient levels of gene delivery in patients.

However, promising preclinical data include the recent development of new therapeutic targets, new treatments such as miRNA and new vectors and, in particular, vectors that are more highly selective with greater therapeutic potential. Clinical trials are ongoing in gastrointestinal cancers, as well as in other tumours such as melanoma, brain, head and neck, prostate or ovarian cancers. Even if safety profiles, drug-resistance and efficacy have to be elucidated further, cancer gene therapy may become a new weapon in anti-cancer strategies.

ACKNOWLEDGEMENTS: Declaration of personal interests: KJ Harrington has received unrestricted educational grants in support of laboratory and clinical research from Oncolytics Biotech Inc. and Genelux GmbH. JP Galmiche has served as a speaker and a consultant for AstraZeneca and Given Imaging. Declaration of funding interests: Y Touchefeu has received a doctoral research grant from the Institute National du Cancer. Part of this work was undertaken in The Royal

Marsden NHS Foundation Trust that receives a proportion of its funding from the NHS Executive; the views expressed in this publication is those of the authors and not necessarily those of the NHS Executive. This work was supported by the Institute of Cancer Research, and Cancer Research UK Section of Radiotherapy (CRUK) Grant Number C46 ⁄ A10588. We acknowledge NHS funding to the NIHR Biomedical Research Centre. We also acknowledge support from the Institute National de la Santé´ et de la Recherché Me´dicale (INSERM).

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     How to cite this article:

     Sree KR and Hanumanaik M: Gene Therapy, Recent Development and Future Prospects in Gastrointestinal Oncology: A Review Article. Int J Pharm Sci Res. 2013; 4(2); 558-574.

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Gene therapy, vectors, genetic diseases, principle of gene therapy