Cancer is a complex disease where normal cells of the body loose their growth regulation resulting in uncontrolled growth and proliferation. Research in cancer deals mainly with its origin, biology and treatment. The incidence of head and neck cancers in India is the highest comprising almost 40% of all the cancers, of which 10% are oral cancers (Bhatacharjee et al., 2006). Such a high incidence is partly attributed to the widespread tobacco and alcohol exposure with a large number belonging to lower socioeconomic strata of the population of India.
Most common type of oral cancer is squamous cell carcinoma which is of epithelial origin. Currently surgery and radiotherapy are standard treatments for patients of HNSCC. Although the treatment of early stage tumors is very effective, most of the HNSCC patients presented to the clinic are at a very advanced stage of the disease. The results of these treatments for advanced cancers are generally poor. The incidence of failure is as high as 60% and the systemic metastasis is seen in more than 20% of the patients (Hill & Price, 1994). Five year survival of patients undergoing radiotherapy is as low as 25% while it is 20% for surgery (Hofele et al., 2002).
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Therefore, conventional therapies including surgery, radiation and chemotherapy are ineffective against such tumors and outcome remains consistently poor, with low survival rate. These drawbacks of the conventional therapies compel for research into new treatment regimes, such as gene therapy. Gene therapy is the latest treatment modality, which has shown promising results in recent years. Several gene therapy clinical trials for cancer as well as other diseases are already underway and some of them have been completed with successful results.
1. Gene therapy
Gene therapy is the alteration, insertion, or deletion of genes from a cell with an aim to treat disease. The principle of most common form gene therapy involves gene transfer in order to correct genetic defects or to express therapeutics within or near target cells. Last two decades have seen gene therapy for cancer treatment evolve with promising results, but still a major hurdle is the transfer of genetic material into the host cell. Efficient delivery of the gene of interest into the host cell depends on the kind of vector employed for this purpose. There are various gene delivery vehicles available for somatic gene transfer with respective advantages and disadvantages which can be divided into two categories viz. viral and nonviral vectors (Pfeifer et al., 2001).
1.2. Viral vectors
Viral vectors are frequently used to transfer genetic material into a host cell. Viruses can efficiently transport their own genome into a host cell and use the host cell's machinery for their own reproduction. Due to this characteristic of viruses they have been considered as a useful tool to deliver a therapeutic gene into a diseased tissue. Viral vectors commonly used for gene delivery are:-
A. Integrating viral vectors
Retrovirus (murine leukaemia virus) - (+)ssRNA virus
Adeno-associated virus - ssDNA virus
Lentivirus - (+)ssRNA virus
B. Non-integrating viral vectors
Adenovirus - dsDNA virus
Alphavirus ÂÂ- (+)ssRNA
Herpes simplex virus - dsDNA virus
Vaccinia virus - dsDNA virus
Some of the most frequently used viral vectors are discussed below:
1.2.1. Retroviral Vectors
Retroviruses are lipid-enveloped particles consisting of a linear, positive-sense, single-stranded RNA genomes of 7 to 11 kb. The RNA genome of the virus is reverse transcribed into linear double stranded DNA after its entry into target cells and then integrated into the host cell chromatin. This family of viruses includes several varieties which have been extensively used in gene therapy: the mammalian and avian C-type retroviruses, lentiviruses (e.g. HIV) and spumaviruses. Retroviruses have long terminal repeat (LTR) sequences at either ends. LTR and the adjacent sequences act in cis during viral gene expression, packaging, retro-transcription and integration. The gag, pol and env genes encode the structural proteins, nucleic-acid polymerases/integrases and surface glycoprotein, respectively. (Kay et al., 2001)
For gene therapy purposes, the retrovirus is modified by replacing the gag, pol and env gene with the cDNA of interest while the packaging signal (Ïˆ) is retained in the viral genome. The gag, pol and env genes are supplied in trans by the packaging cell line that is transfected with these genes. Upon transfection of the modified retrovirus genome into these packaging cells, they serve as virus producing cells (VPC). Most commonly vectors are generated from Moloney murine leukemia virus (MoMuLV) in which up to 8-kb of exogenous DNA can be inserted and expressed in place of the viral genes.
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Implantation of vector producing cells has been shown to effectively eliminate tumors in animal models. In a report by Ambade et al., the implantation of HSV-tk vector producing cells followed by IL-2 therapy resulted in an efficient in vivo transduction and regression of HNSCC tumor xenograft (Ambade et al., 2010). In a similar study, Walling et al., reported efficient bystander-mediated regression of osteosarcoma via retroviral transfer of the HSV tk and IL-2 genes (Walling et al., 2000). In another study Zeng et al., directly injected HSV-tk carrying retrovirus vector into breast cancer xenografts and showed tumor regression (Zeng et al., 2006).
Although retroviral vector mediated gene therapy has shown promising results, one of the major disadvantages is that the successful transduction by retroviral vectors is strictly dependent on target cell mitosis shortly after entry. At any given time only a fraction of cells pass through mitosis this severely limits the applications of retroviral vectors in gene therapy (Kay et al., 2001). Another major shortcoming with the retroviral vectors is that they integrate into the host genome thereby increasing chances of insertional mutagenesis. However, some encouraging clinical results of gene therapy have been obtained with these vectors. (Barquinero et al., 2004 and Deisseroth et al., 1999)
1.2.2. Adeno Associated vectors
Adeno-associated viruses (AAVs) are human parvoviruses that usually require a helper virus, like adenovirus, to achieve a successful infection. AAVs are not known to cause any disease but were initially discovered as a contaminant in an adenovirus preparation. There are six known human viral serotypes but the most commonly used serotype for gene therapy is AAV-2 especially because no known disease is associated with its infection.
The viral genome consists of two genes, rep which is required for viral genome replication; and cap which encoding structural proteins. Flanking these genes are Inverted Terminal Repeats (ITRs) that are 145 nucleotides long. Each particle contains a single plus- or minus-strand genome. For producing functional AAV vectors, structural (cap) and packaging (rep) genes are replaced by the therapeutic gene. cap and rep genes can be supplied in trans through the helper adenovirus however, ITR sequence is required in cis and therefore is retained in the AAV vector backbone. AAV vectors can either randomly integrate into the host chromosome or can be maintained episomaly (Kay et al., 2001)
AAV-mediated gene transfer of HSVtk has been reported to sensitize human oral squamous cell carcinoma cell lines to ganciclovir (Fukui et al., 2001). Intratumoral injection of AAV has been demonstrated to show anti tumoral effects. Li et al. demonstrated enhanced anti tumor effects on recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer. They showed that GCV treatment of infected MCF-7 cells under the Dox induction had more inhibitory effects than those without Dox induction. Tumor growth of BALB/C nude mice breast cancer was also retarded after rAAV-2/HSVtk/Tet-On injection into the tumors under the Dox induction (Li et al., 2006).
The packaging capacity of AAV is about 5.0 kb, which is a major limitation of this vector system. However, clinical trials using AAV for the treatment of cystic fibrosis, hemophilia and muscular dystrophy are underway. (Kay et al., 2001)
1.2.3. Adenoviral Vectors
Adenoviral vectors are one of the most commonly used viral vectors in clinical trials today as compared to Retroviruses which are the second most commonly used vectors. According to the Journal of Gene Medicine approximately 25% of the clinical trials today are using adenoviral vectors as a mode of gene delivery.
Adenoviruses are dsDNA viruses with a size of approximately 36 kb. Adenoviruses have several features that make them well suited for use in gene therapy.
Adenoviruses are ubiquitous and more than 100 different serotypes are known with approximately 43 serotypes isolated from humans. Most adults have been exposed to the adenovirus serotypes 2 and 5 which are frequently used in gene therapy.
Adenoviral vectors show a broad tissue tropism i.e. they can infect a broad range of cells with transduction efficiency higher than the other currently used vectors. Adenoviral vectors have low pathogenicity in humans with mild symptoms associated with the common cold.
Adenoviral vectors can accommodate up to 7.5kb of insert DNA and can infect both dividing as well as non dividing cells.
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Since the viral genome does not undergo a lot of rearrangement, the inserted foreign genes is usually maintained without any change through successive rounds of viral replication. No insertional mutagenesis is observed.
Adenoviral vectors can be easily manipulated using recombinant DNA techniques.
Adenovirus enters the host cell via receptor mediated endocytosis. Coxsackie and adenovirus receptor (CAR) interacts with the adenovirus fiber and mediates internalization of virus into the host cell. In addition to CAR, Î±v integrins act as coreceptors and aid viral endocytosis. To increase the safety measurements of adenoviral vectors in gene therapy, most adenoviral vectors are made replication incompetent by deleting the E1A region of the adenoviral genome responsible for replication of the adenovirus. The E3 region of the adenoviral genome is also deleted which renders the virus non pathogenic and also increases the space in the genome available for the insertion of transgenes (Gommans et al., 2005).
4.3. Suicide Gene Therapy
Suicide gene therapy, also known as enzyme prodrug-therapy, involves the delivery of a gene encoding a foreign enzyme (usually of viral or bacterial origin) into a target tissue and upon administration of a prodrug; the encoded enzyme can convert the prodrug into a toxic form and can bring about cell death. Suicide gene therapy basically includes two components viz. the prodrug and the suicide gene. The ideal suicide gene should exhibit certain characters: (Morris et al., 2002)
The expression of the suicide gene, in itself i.e. in the absence of the prodrug; should not be toxic
The enzyme should be able to rapidly activate the prodrug,
It should work efficiently at low prodrug concentrations,
The gene should encode a relatively low molecular weight protein due to size restrictions of commonly used gene transfer vectors, and
The encoded enzyme should preferably be a monomeric protein to avoid the need to introduce more than one coding sequence into the vector or having the appropriate relative expression of each subunit to achieve a functional enzyme.
Characteristics of the ideal prodrug include:
Lack of prodrug mediated toxicity in the absence of expression of the suicide gene
Use of a clinically approved prodrug
A long half-life of the activated prodrug and
A drug with a "bystander effect" is usually desirable, at least for use in cancer treatment.
4.3.1. Herpes Simplex Virus Thymidine Kinase (HSV-tk)/Ganciclovir (GCV)
The herpes simplex virus-1 thymidine kinase is the most common suicide gene used in gene therapy. Though the natural substrate for this enzyme is thymidine (Pilger et al., 1999) it also has a high affinity for nucleoside analogues like ganciclovir (GCV) and acyclovir (ACV). Compared to the mammalian thymidine kinase, the HSV-Tk has a 200 fold higher activity for monophosphorylation of these drugs into their GCV-MP or ACV-MP forms. The mammalian thymidine kinase then converts the monophosphorylated forms into di- and tri- phosphorylated form. Upon addition of GCV, HSV-tk expressing cells accumulate the toxic GCV-phosphate and subsequently are killed via different mechanisms like inhibition of DNA polymerase and DNA chain termination by incorporation of GCV phosphate into the DNA chain. (Xu et al., 2001, Morris et al., 2002). However, sensitivity of cells to HSV-tk/GCV may also depend on the level of DNA synthesis and on the activity of cellular kinases that convert the monophosphorylated form into the di- and tri-phosphorylated product. Apart from GCV, acyclovir and oral penciclovir have also been extensively used in clinical trials.
One of the major advantages of suicide gene therapy is that the toxic metabolite - triphosphorylated GCV can be transferred to adjacent non transduced cells. This phenomenon is commonly known as 'bystander effect'. Hence, not all cells need not express HSV-tk in order to be killed by the HSV-tk GCV system. The toxic metabolite can be transferred across neighboring cells via gap junctions or by phagocytosis of apoptotic vesicles containing GCV-phosphate released from surrounding tumor cells. It is also suggested that the antitumor immune mechanisms in response to tumor necrosis may aid the bystander effect (Morris et al., 2002).