Gene delivery vehicles incorporated in Gene
Therapy for breast cancer: current technology and potential options
Abstract
Breast cancer has affected many women all over the world. The use of gene therapy for the treatment of breast cancer is being widely researched. Gene therapy is the insertion of genetic material, known as gene delivery vehicles, into the targeted/infected cells so as to alter the phenotype of the target cell (Chen et al. 1995). Gene therapy methods can be broken down into four categories; (1) the insertion of suicide genes, (2) the use of DNA plasmids, (3) decrease tumour progression and increase immunogenicity (4) use of stem cells along with chemotherapy. The gene delivery vehicles used for these therapies are adenoviral vectors, lipofection, non viral vectors, retroviral vectors, bone marrow stem cells and vaccines. Although there is a huge variety of gene delivery vehicles not all of them are efficient in the proliferation of tumour cells and tissues. One of the main limitations of these vehicles is their specificity. Their lack of specificity tends to expose, normal uninfected cells to toxicity. This means that to prevent the normal cells from dying even if this particular gene therapy was efficiently working it would not be used clinically because it could kill the patient. Further research is being undertaken to deliver genes with specific tumour/tissue promoters which can guide the genes to their particular targets. Gene therapy is the current technology being used in the treatment of cancer but unfortunately it does not guarantee the permanent removal of the breast cancer cells but it does increase the survival rate by inhibiting growth of the tumour cells.
Introduction
People all over the world are prone to cancer. They may acquire the cancer through certain gene or protein mutations or certain environmental factors. From all forms of cancer breast cancer is one cancer which affects women in a deadly manner. Even though breast cancer like other cancers can be caught at an early stage that does not guarantee that the cancer will not show up again. Gene therapy is one therapeutic method which is being used widely to target the cancer cells and prevent them from resurfacing. Gene therapy is the transfer of suitable genetic material into a specific cell type, which could be a solid tumour or the host cell, so as to alter the phenotype of the target cell (Chen et al. 1995). Gene therapy does not involve only one form of gene delivery vehicle it takes into account different genes and proteins which could treat the cancer. Widely used gene delivery vehicles in gene therapy are; adenoviral vectors, which are viruses, and ligand-liposome, which are non viruses. Each of these categories consists of different types of genes and proteins being used for their delivery in the treatment for cancer. Adenoviral vectors are the most commonly used gene delivery vehicles but they do hold a limitation in cancer therapy. When delivered to the cells adenoviral vectors are nonspecific therefore transduction of therapeutic genes in non target cells, which are the normal uninfected cells, because this sort of gene delivery vehicle carries strong antiviral immune responses which along with eliminating the virus can also cause death of the patient (Chen et al. 1995; Janát-Amsbury et al. 2004). In the case of lipofection, which is a non viral vector, is that it lacks an efficient tumour-targeting system. Hence even though ligand-liposome has the ability to be modified into the perfect delivery vehicle its lack of specificity can be very toxic to the rest of the cells (Rait et al. 2002). In conclusion the most common delivery vehicles have the same limitation but what sets them apart is what form of gene is used and also if other treatments are involved along with the gene delivery method, in this case chemotherapy, promoters, DNA plasmids etc. for the treatment of cancer.
The purpose of the following review is to therefore examine the different types of gene therapies used along with different gene delivery vehicles. The review also discusses which of these therapies have been used clinically for the treatment of cancer and also the role they play alongside other major treatments. In addition the review provides a look into the further research being undertaken so as to improve the methods of gene therapy.
Background
Gene therapy methods and associated gene delivery vehicles.
Gene therapy methods used for the treatment of breast cancer can be placed into different categories;
(1) the insertion of suicide genes,
(2) the use of DNA plasmids,
(3) decrease tumour progression and increase immunogenicity
(4) use of stem cells along with chemotherapy.
The insertion of suicide gene therapy involves the transfer of drug-activated enzyme into the effected tumour cells and the cell is then treated with a prodrug form of chemotherapeutic agent that increases the concentration of the drug in the tumour following the apoptosis of tumour cells (Takahashi et al. 2006). (
1) Suicide gene therapy uses human adenoviruses as gene delivery vehicles. Human adenoviruses are DNA viruses of genomic size of 36 kb and are not surrounded by a membrane (Chen et al. 1995).
(2) The advantage of using DNA plasmids for gene therapy is their unique feature which allows them to be delivered without detecting the existing tumour. Therefore the tumour can be suppressed significantly even if it isn’t detected before delivering the DNA plasmid and this therapy decreases small tumours present in internal organs (Horton et al. 1998).
(3) Breast cancer and other cancers all consist of tumours which are the target tissues, therefore to treat cancer the tumour needs to be suppressed. Unfortunately tumours cannot be completely removed but gene therapy allows an immune response to be brought about in the target tissues. This immune gene therapy uses the transfer of cytokine genes to enhance immune response, co-stimulatory molecule genes and antigen molecule genes (Takahashi et al. 2006).
(4) The popularity of gene therapy has increased over the years because of its ability to be used alongside chemotherapy for more efficient treatment. The use of bone marrow stem cells along with chemotherapy as a delivery vehicle not only produces rapid hematopoietic recovery but at the same time is free of toxicity. Therefore normal uninfected cells will not be destroyed in this process (Koc et al. 2000).
Specific genes involved in gene therapy
There are a number of genes which are used as delivery vehicles but not all of them are successful for the treatment of cancer. The first category of gene therapy which involves the insertion of suicide genes showed a breakthrough clinical trial which used replicated human adenoviruses along with a DF3 promoter to express β- galactosidase and the herpes simplex virus in thymidine kinase gene in DF3 breast carcinoma cells (Chen et al. 1995). Thymidine kinase gene (HSV-tK) DF3 antigen are members of high molecular weight glycoproteins which are over expressed in breast carcinoma cells (Chen et al. 1995). This suicide gene when transferred encodes and activates an enzyme that can then activate a prodrug within the tumour cells so as to leave the tumour cells susceptible to other agents leaving the normal cells non toxic to these agents. Another type of gene used is Her-2 ODN. Her-2 ODN like thymidine kinase is over expressed in the tumour cells. Her-2 ODN is delivered to the tumour cells through cationic liposomes. These liposomes are considered to be attracted for gene delivery because they can easily be modified, prepared and have low toxicity. Cationic liposomes are composed of positive charged lipid bilayers and can be modified to negatively charged ODN’s by the process of mixing which results in an overall positive charge. This complex is called folate- liposome- AS Her-2 ODN. This complex inhibits MDA-MB-435 xenograft growth in aggressive breast cancer cells even though Her-2 ODN is not over expressed in them by making MDA-MB-435 more sensitive leaving them prone to suppression, inhibition and also sensitive to toxic treatments like chemotherapic agents and radiation while at the same time preventing the normal cells from these toxic agents (Rait et al. 2002; Jhaveri et al. 2004).
The second category of gene therapy method is the use of DNA plasmids as gene delivery vehicles. This is a cancer treatment which does not involve local injection or identification of existing tumour nodules. This cancer treatment injects plasmid DNA (PDNA) encoding murine interferon α (mIFN-α) which provides anti tumour effects on primary and metastic tumours. The advantage of this delivery system is that tumours do not have to be identified prior to the injection of the plasmid which means its target is specific (Horton et al. 1998). Along with interferon α interferon β can also be used in effective treatment of solid cancer tumours. IFN-β gene is directly injected into the tumour which showed a substantial deterioration of the tumour. The effects portrayed can be autocrine or paracrine because the cells secrete the gene. Small amount of IFN-β gene along with defective replicated adenoviral vector can reduce the size of the tumour and can also inhibit tumour growth. It shows a direct antiproliferative or cytotoxic activity of IFN-β gene. This form of delivery system can also be used along with chemotherapy for more advanced treatment. (Qin et al 1998)
The third category of gene therapy method is the decrease in tumour progression and increase immunogenicity. This certain gene therapy not only takes into consideration the use of adenoviral vectors but also vaccine virus vector. In this case a multifunction DNA vaccine is used. In addition to this gene IL-18 enhances cellular mechanisms by up-regulating MHC class I antigen expression and activating the T cells to bring about a cellular response. They also affect the tumour angiogenesis sufficiently inhibiting the function of the tumour in turn suppressing it. DNA vaccine encodes polyubiquitanation transcription factor Fra-1 and secretory IL-18 which prevents primary breast tumour growth and metastases by suppression of tumour angiogenesis and activation of T, NK, and dendritic cells.
Polyubiquitanation is a process which makes many copies of this cellular protein to covalently attach to a target protein so as to increase the chances of degradation by proteasome (Luo et al. 2003). Another gene delivery system used is a non viral gene delivery system and a propapoptotic gene, bik. bik gene is a proapoptotic member in the Bcl-2 gene family. The bik gene is a potent inducer for apoptosis. p53 is a gene mutation which results into cancer. The gene does not require the function of p53 and hence can perform apoptosis. Apoptosis is a form of programmed cell death. In the study which attempted to use this gene along with non viral delivery system significantly inhibited the growth and metastasis of the human breast cancer cells allowing an increase in the life span of the patient, in this case the mice (Zou et al. 2002) Another non viral gene delivery system has also brought about successful treatment which is administrated towards prevention of cancer. IL-12 which comes from the same family as IL-18 plays a factor in overcoming the suppression of T cells. The IL-12 gene delivery was combined with paclitaxel chemotherapy to bring about a substantial decrease in the tumour and at the same time producing an antitumoral effect without causing any release of toxicity. IFN-g (interferon g) which is formed after the induction of T and NK cells activates macrophages to produce NO (nitric oxide) directly killing the tumour cells (Jana´t-Amsbury et al. 2004). One of the most efficient gene delivery vehicles are adenoviruses. Combining an adenovirus and adeno-associated virus allows the transfer and expression of antisense molecules in tumour cells. This type of hybrid vector retains advantages of bith virus vectors by consisting of high transfection efficiency along with the capacity to incorporate exogenous DNA into a hosts DNA. This delivery system suppresses the tumour and also brings about the effect of apoptosis of the human breast cancer cells. Along with gene delivery vehicles, anticancer therapy can also be used. For example a study was conducted to prevent the over expression of the protein Rad51 in breast cancer cells. The study designed a Rad51 promoter based anti cancer therapy. The promoter allowed only the malignant cells to be removed leaving the normal tissues undamaged. This type of therapy also falls into gene therapy because it is using proteins and Rad51 is a major component of recombination so advancement in anti-cancer therapy can also increase immunogenicity while decreasing the tumour progression (Hine et al. 2008).
The fourth category is the use of stem cells for gene therapy. Mesenchymal stem cells (MSC) produce adeventitial cells in the marrow environment provide support to hematopoiesis by producing membrane-bound and soluble signals and cytokines. MSC’s enhance hematopoietic engraftment rate and quality after myeloablative and stoma-damaging treatments (Koc et al. 2000). Another form of stem cells extensively used are AC133+ progenitor cells (APC) can act as both gene delivery vehicles and cellular probes for magnetic resonance imaging (MRI). The experiment conducted used superparamagentic iron oxide (SPIO)-labelled APCs to carry the gene human sodium-iodide symporter to the sites of the tumour. This allowed multiple imaging modalities to observe the migration and accumulation of stem cells (Rad et al. 2009). Another gene which plays an important role in the treatment of cancer and gene therapy is BRAC1. This gene is developed from the mammary stem cells. This gene can explore early cellular changes that occur in BRCA1 mutation carriers (Lim et al. 2009).
Discussion
Limitations
Gene therapy for cancer is becoming more advanced as new genes are being discovered which can assist as gene delivery vehicles. But like every other treatment gene delivery vehicles do have certain limitations which prevents them from providing a complete treatment for cancer. One of the major limitations is the lack of selective delivery of adenoviruses or lipofection to tumour cells. Generally only a small fraction of the administrated dose of the virus reaches the site, whereas the rest of it gets distributed throughout the body resulting in damage to normal tissue. Poor tumour transduction and non-specific cell infection are also factors limiting the full potential of gene therapy. Therefore all known gene delivery systems have the same limitation which is specificity (Bidwell and Raucher 2005; Qin et al. 2002; Stoff-Khalili 2005 and Trimboli et al. 1999).
Other therapies
There are other gene therapies used in the past which haven’t been very successful. In the case of non viral delivery system a DNA complex mediated gene transduction through receptor endocytosis have been reported to have delivered genes. But this approach was very limiting because it lacked transduction efficiency. Retroviral vectors have also been used extensively but haven’t been successful because of their low viral titters and dependence on target cell replication (Chen et al. 1995; Zou et al. 2002). During the past several years peptides have been developed that inhibit various oncogenes. This therapy still needs to be researched further but does show potential (Bidwell and Raucher 2005). Bone marrow stem cells are likely to improve hematopoiesis after myelotoxic treatment. One of the revised treatments which could be tested is the infusion of autologous culture-expanded MSCs along with PBPCs may improve the bone marrow microenvironment and, subsequently, the rate and quality of hematopoietic recovery in heavily pre-treated chemotherapic patients (Koc et al. 2000).
Further Research
The gene therapies described throughout the review are just few of the popular treatments used for cancer. This does not mean that other therapies which are being experimented on and studied are not useful. Adeno viral vectors hold substantially more potential than lipofection, retroviral vectors and non viral vectors. They do administer specificity but are considered to be toxic to the normal cells. But alongside chemotherapy or radiation these gene delivery vehicles will be able to help in the treatment of cancer faster and at the same time increase the survival rate. Further research is still being conducted so as to perfect the gene therapies which have been tested. It has been concluded that because non viral vectors and adenoviral vectors have limited specificity certain tissue/ tumour specific promoters can be used. Hence this provides a guide for these delivery systems to the target cell while preventing normal cells from exposure to toxicity (Zou et al. 2002).
Conclusion
Gene therapies are evolving over the years and new technology has been introduced but more research and clinical trials need to be performed. The reason for this is that even though these therapies can prevent breast cancer from spreading and significantly reduce tumour growth it does not guarantee the full removal of the breast tumour and cancer cells. The gene therapies mentioned in the review have undergone clinical trials but they all have the same limitation, specificity. In conclusion more gene delivery vehicles need to be discovered in order to improve specificity and at the same time cure cancer permanently.
