Authors: Nicole Lindsay-Mosher, Cathy Su
Institution: McMaster University, University of Toronto
Cancer causes one in seven deaths worldwide, making it one of the most important issues in the world of biotechnology today. Current cancer therapies, including chemotherapy and radiotherapy, have severe side effects and often prove ineffective at completely eradicating malignant cells. Therefore, a more selective method of targeting tumour cells must be designed. Gene therapy holds great potential to selectively target cancer cells, allowing the treatment to effectively destroy the cancer while leaving healthy tissues intact. In order to develop a gene therapy treatment, two main obstacles must be overcome: a therapeutic agent must be developed to facilitate genetic changes, and a delivery method must be optimized to insert the therapeutic agent into target cells. Recent advancements in both the design of the therapeutic agent and the delivery method allow changes in both the genome and in gene expression to be performed in the target cells with a high degree of accuracy and efficiency. This review highlights several evolving technologies currently being developed for gene therapy, as well as strategies that could be employed using these technologies to treat cancer. Although not currently in widespread use, gene therapy is extremely promising as a treatment for cancer.
Cancer is estimated to cause about one in seven deaths worldwide (American Cancer Society, 2016). This disease is characterized by the transformation of healthy tissue into malignant and invasive tumour tissue that rapidly divides and takes up resources needed by other cells (Hanahan & Weinberg, 2011). Although effective treatments for some forms of cancer are available, losses are astounding; in 2012 there were over 14 million new cases of cancer, and 8.2 million cancer related deaths (Siegel, Miller, & Jemal, 2015).
There are hallmarks common to cancer tissue which can be used to differentiate malignant cells from healthy ones. These include dysregulation of the cell cycle, cell growth and differentiation, and apoptosis (Hanahan & Weinberg, 2011). Cancer spreads between different parts of the body after the earliest stages in a process known as metastasis (Hanahan & Weinberg, 2011). Metastasis adds a layer of complexity to treatment because metastatic cancers are both mobile and heterogeneous. One of the greatest challenges in treating cancer is the extreme heterogeneity of the disease; different types of tumours often display drastically different genetic and phenotypic characteristics. This heterogeneity ultimately makes cancers highly resistant to almost all forms of treatment (Hanahan & Weinberg, 2011).
The goal of cancer therapy is to specifically eliminate cancerous tissue while minimally impacting healthy tissue, and to maintain the effects of therapy over time without harmful side effects. Currently, common treatment options for cancer include chemotherapy, radiotherapy, and surgery. Chemotherapy uses chemical agents to target cells undergoing DNA replication, a strategy which effectively kills rapidly proliferating cells (Urruticoechea et al., 2010). Radiotherapy also targets rapidly growing cells, but uses radiation rather than chemical agents to kill tumour cells. However, tumour cells often prove resistant to chemotherapy and radiotherapy due to their increased viability and growth rate relative to healthy cells (Kaliberov & Buchsbaum, 2012). Surgery involves the removal of malignant tissues, an approach which is very effective at removing large solid tumours but is often limited in treating small or metastatic tumours (Urruticoechea et al., 2010). Clearly, there is a need for new cancer treatments which are highly effective at targeting cancer cells, while being selective enough to leave healthy cells untouched.
Unlike chemotherapy and radiotherapy, gene therapy has the potential to target tumour cells with a high degree of accuracy. However, current gene therapies to treat cancer are still in experimental stages. There are many technologies available and it can be confusing to distinguish between them and evaluate the different strategies. This article will review progress in the gene therapy field along with potential applications to cancer.
Genetic Basis of Cancer
The abnormal regulation of cancer cells is caused by mutations in two classes of potentially harmful genes: proto-oncogenes and tumour suppressors. Proto-oncogenes become cancer-causing oncogenes through ‘gain-of-function’ mutations which increase the impact of the gene to harmful levels by, for example, creating a constitutively active protein product. 'Loss of function' mutations in tumour suppressor genes also facilitate cancer development, as they prevent the gene from producing enough of a useful protein product that contributes to growth regulation. Tumour suppressor genes perform functions such as arresting cell division or initiating apoptosis. As such, loss-of-function mutations in tumour suppressor genes contribute to the uncontrolled proliferation observed in cancer (Hanahan & Weinberg, 2011).
Gene therapy can be used to target both oncogenes and tumour suppressor genes. Treatments for the former seek to counter or inactivate gene expression and to lower gene product formation, while treatments for the latter seek to restore gene expression. Gene therapy has been successful in treating genetic diseases with single gene defects, including immune disorders (e.g. Wiskott-Aldrich syndrome), blood cell disorders (e.g. beta-thalassaemia) and metabolism disorders (e.g. X-linked adrenoleukodystrophy) (Braun et al., 2014; Cartier et al., 2012; Malik & Arumugam, 2005). Gene therapy can be targeted to both germ and somatic cells, meaning that it is possible for genetic alterations to be passed on to children.
Gene Therapy Strategy
In order to modulate the expression of oncogenes and tumour suppressors, two main challenges must be overcome. The first challenge is to create a therapeutic agent which alters gene expression, and the second challenge is to deliver the desired therapeutic agent to the target cells. In the early years of gene therapy, both of these challenges were met in a trial that tested the use of a retroviral delivery method of a BRCA1 tumour suppressor gene splice variant to treat breast cancer (Tait, Obermiller, Hatmaker, Redlin-frazier, & Holt, 1999). During Phase I clinical trials, patients showed tumour suppression with highly effective gene transfer and little immune response, particularly in small tumours. However, Phase II patients showed no response to this form of therapy, because they had developed antibodies against the retroviral envelope (Tait et al., 1999). This immune response is a common shortfall of retroviral vectors (Wu & Dunbar, 2011).
Another well-known study used bone marrow cells transduced with a retrovirus containing a therapeutic gene to treat X-linked immunodeficiency (SCID-X1). Although immune cell count and function seemed to have been normalized in the adolescent patients, four of the nine treated patients developed leukemia in later stages of the treatment (Hacein-Bey-Abina et al., 2008). The cause of this drastic side effect was found to be retroviral-mediated insertion of the therapeutic γC cytokine receptor into an unintended target, which altered a proto-oncogene involved in T cell self-renewal and differentiation (McCormack et al., 2010). Since regulation of T-cell fate was disrupted, the treated cells started to multiply uncontrollably, ultimately leading to cancer.
In order to avoid unintended effects, new methods of gene therapy must target pathogenic mutations in a highly specific manner. Unlike the retroviral insertion strategy used in the SCID-X1 trials, therapeutic genes must be delivered to a specific area of the genome or transcriptome. As gene therapy progresses, safer and more effective choices of therapeutic agents and delivery methods must be explored to avoid the shortfalls of viral vectors as outlined above. Determining the appropriate gene targets will require a more comprehensive understanding of different tumours at a molecular level, a challenge which is outside of the scope of this review. This article will focus on possible approaches to combat cancer once an important proto-oncogene or tumour suppressor gene has been identified. This review covers several promising methods of introducing or silencing target genes once they have been identified, as well as how these methods may be used in combination to optimize the effectiveness of the treatment.
Although the field of gene therapy has advanced rapidly in the past few decades, there are several issues still hindering the development of effective gene therapy treatments. These problems include low efficiency of gene transfer, failure to deliver genes larger than 5kb in size, regulation of transgene expression in the host cells, and ineffectiveness against autosomal disorders (Yang & Walsh, 2005). This review aims to explain some of the most useful components of the gene therapy toolkit that are applicable towards treating cancers, and catalogue their strengths along with their shortfalls.
TOOLS FOR GENE THERAPY
Part 1: Designing the Therapeutic Agent
The first challenge after identifying the target gene to be used for gene therapy is to construct a therapeutic agent to alter the expression of the gene of interest. This therapeutic agent must be designed with three main concerns in mind: specificity, efficiency, and transiency. Specificity refers to the ability of the therapeutic agent to alter the expression of the target gene in the target cells without causing undesirable mutations in other parts of the genome or in healthy tissues. Efficiency is primarily concerned with the ability of the therapeutic agent to modify the genome or gene expression at a low dosage. Another important factor is the transiency of treatment, i.e. how long the alterations in gene expression will last in the patient. There are advantages and disadvantages to both transient and lasting variations of gene therapy, and the choice of therapeutic strategy should be informed by the individual case; specificity and efficacy, on the other hand, should always be maximized in order to give the best possible outcome.
SmaRT: Gene Silencing through Pre-mRNA
Spliceosome-mediated RNA trans-splicing (SmaRT) is a technique which corrects mRNA after transcription. Trans-splicing occurs through ligation of exons from different transcripts. Cis-splicing, on the other hand, refers to the conventional eukaryotic mechanism of splicing of a single transcript. Pre-mRNA trans-splicing molecules (PTMs) can be designed to carry a binding domain that targets a specific intron in the normal pre-mRNA (Yang & Walsh, 2005). Together with the spliceosome, the PTM can cause trans-splicing of pre-mRNA with up to 80% efficiency as compared to cis-splicing (Yang & Walsh, 2005). It can be used to repair the mRNA transcripts of aberrant genes, but the utility is that the effects are relatively reversible. However, designing the PTM is not easy. One approach is to randomly generate a collection of sequences, then use fluorescence-activated cell sorting (FACS) of cells containing these sequences to identify the optimal construct (Yang & Walsh, 2005). In this case, each PTM has a portion of a gene which encodes the green fluorescent protein, and FACS allows for selection of successful trans-splicing.
TALENs and ZFNs: Site-Specific Modification
While SmaRT targets pre-mRNA or mRNA and is therefore reversible, other methods of gene therapy cause more permanent alterations by modifying the genome itself. Chimeric nucleases are molecules composed of a sequence-specific domain fused to a nonspecific DNA cleavage molecule, and are capable of inducing double-strand breaks (DSBs) at specific sites in the genome (Gaj, Gersbach, & Barbas, 2013). Unlike SmaRT systems, chimeric nucleases cause permanent changes to the DNA of the target cells, which persist long after expression of the nucleases themselves has ceased.
Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are two types of chimeric nucleases, each with a distinct class of customizable sequence-specific domain (Gaj et al., 2013). ZFNs have multiple zinc finger domains, structural motifs containing zinc ions, and a conserved ββα motif. Residues on the surface of the α-helix bind to three base pairs on the major groove of DNA, allowing each zinc finger domain to recognize a nucleotide triplet. ZFNs contain an array of zinc finger domains which together can recognize a specific sequence (Gaj et al., 2013). Each zinc finger domain recognizes a specific nucleotide triplet rather than a single base pair. As a result, the zinc finger array can bind to certain sequences with high specificity; however, only certain nucleotide triplets have corresponding zinc finger domains. Therefore, the number of sequences to which ZFNs can bind is limited (Osborn et al., 2013). TALENs, by contrast, are composed of an array of amino acid repeat domains, each of which recognizes only a single base pair. Therefore, TALENs can be engineered to recognize any DNA sequence (Osborn et al., 2013). However, TALENs are more expensive to make than ZFNs because the amino acid repeat domains are complex and difficult to synthesize (Gaj et al., 2013).
Both ZFNs and TALENs can be used to induce DSBs at specific loci with similar frequencies (Hockemeyer et al., 2011). The creation of DSBs can be used to excise sequences from the genome or to insert donor DNA. To remove a sequence (e.g. a harmful oncogene), DSBs can be induced at either end of the sequence and the free ends ligated, eliminating the DNA between the DSBs. To insert a sequence (e.g. a helpful tumour suppressor), the desired DNA sequence can be introduced to the cell along with a chimeric nuclease that makes a single DSB, allowing the donor sequence to be ligated into the DNA. These methods lead to specific and long-lasting genetic modification (Hockemeyer et al., 2011). However, TALENs and ZFNs are both extremely time- and resource-intensive.
Viral Vectors: High-Level Transgene Expression
Viruses proliferate by using the cellular machinery of host cells for the expression of viral proteins and replication of the viral genome. Engineered viruses present an efficient delivery system (see ‘Delivery Method’ below) as well as a high level of expression of transgenes (Waehler, Russell, & Curiel, 2007). Viral vectors can be designed by replacing portions of the viral DNA with the transgene of interest coupled with a promoter which will allow the transgene to be expressed once inside the host cell (Kootstra & Verma, 2003). To prevent the engineered virus from threatening healthy host cells, all portions of the viral genome which are not essential for infection of the host cells are deleted, including genes encoding the viral capsid and other virulence factors. Viral vectors can be used to induce short-term or long-term expression of transgenes, depending on the type of vector used (Kootstra & Verma, 2003). Retroviral vectors insert transgenes into the host genome, causing long-term expression of the genes. In contrast, adenoviral vectors do not integrate into host DNA, so the viral transgenes are expressed and replicated independently of the host genome. These non-chromosomal pieces of genetic material are referred to as ‘episomes’. Because these episomes degrade over a period of weeks to months (depending on the activity of DNA repair and destruction pathways), adenoviral vectors result in only short-term expression of transgenes (Kootstra & Verma, 2003). Short-term expression of transgenes may be useful in some cases, but cannot permanently correct harmful genetic defects. Long-term genetic modification of the host genome via retroviral vectors is one alternative, but poses significant risks: integration of the transgene into the genome can induce mutations at off-target sites in the genome, a process called ‘insertional mutagenesis’ (Wu & Dunbar, 2011). This can result in harmful side effects, including dysregulation of proto-oncogenes and tumour suppressors, as in the SCID-X1 trials.
CRISPR: Targeted Gene Regulation
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are a bacterial defense mechanism for destroying foreign DNA (Marraffini & Sontheimer, 2010). Currently CRISPR is the most attractive therapeutic agent available for gene editing and silencing in mammalian cells. It remains relatively specific and simple to design compared to other genome editing technologies such as TALENs and zinc finger nucleases. This technology requires only two types of DNA molecules which can be encoded on the same construct and introduced into a variety of hosts: a molecule that encodes Cas (CRISPR-associated system) endonuclease, whose role is to induce a double stranded break, as well as a molecule to encode guide RNA (gRNA) which guides the nuclease to target a specific DNA or RNA element. gRNAs are customizable towards each target, allowing for high versatility. Cas9 is a variety of Cas from the bacteria Streptococcus pyogenes (Wilkinson & Wiedenheft, 2014). Cas9 combined with a small gRNA that targets the promoter region of a gene can cause up to 100-fold repression (Qi et al., 2013). Several recent innovations, including converting Cas9 into a nickase enzyme and using truncated gRNAs, have further increased the specificity of the CRISPR/Cas9 system (Gori et al., 2015). As a result, this technique minimizes the risk of unwanted mutations in the target cells. In addition, the CRISPR/Cas9 system can be used to create targeted modifications at multiple different loci using only one transgene (Dow et al., 2015). The specificity, efficacy and versatility of CRISPR all contribute to the vast potential of this technique in clinical applications.
Part 2: Customizing the Delivery Method
Equally important to selecting the best therapeutic agent out of those available is the task of finding an effective delivery method for introducing each agent. The ideal method must be able to deliver the therapeutic agent to specific tissues where therapy is needed, but must have minimal toxic side effects. The methods explored in this section are summarized in Figure 1.
Liposomes are enclosed phospholipid bilayer structures which can encapsulate oligonucleotides or drugs for delivery. Their size and contents are highly customizable, and they can be targeted to a specific cell type through receptor-mediated endocytosis. Leakage, specificity of targeting, half-life, toxicity versus efficacy (therapeutic index), and success of delivery across the cell membrane are key issues which still need to be overcome in this delivery method (Allen & Cullis, 2013). In order to overcome physiological barriers to access tumours, the ideal liposome would minimize leakage, release its contents only in malignant tissues, have a long half-live in vivo, be non-toxic to healthy cells, and transfect cancer cells with a high degree of efficiency.
Hydrogels are synthetic pockets formed by the assembly or crosslinking of hydrophilic polymers whose pores may be used to carry therapeutic agents. As a delivery system hydrogels can improve vector transfer into specific cells in vivo because they can release their contents at a rate controlled by the speed of diffusion through the hydrogel (Caló & Khutoryanskiy, 2014). Hydrogel encasement increases vector stability, shields vectors from immune effects, and modulates the length and location of vector delivery (Seidlits, Gower, Shepard, & Shea, 2014). Though their versatility is a strength, the exact properties of the hydrogel system must be fine-tuned and tested before each application to ensure maximum efficiency. These features include but are not limited to pore size, hydrogel shape, charge, pH, biomimetic properties, architecture, and degradation time (Caló & Khutoryanskiy, 2014).
Viral Vector Delivery Systems
Pathogenic viruses have evolved to efficiently infect, or ‘transduce’, target cells (Kootstra & Verma, 2003). Viruses can be engineered to deliver therapeutic agents such as toxic genes or CRISPR, a strategy which is discussed later in this review. The transduction efficiency of viral delivery systems is very high compared to non-viral methods of gene delivery such as electroporation or tissue particle bombardment, but viruses also pose significant risks (Kootstra & Verma, 2003). To minimize these risks, it is necessary to target viral delivery systems to diseased cells. They will infect cells based on the tropism, or affinity for a given cell type, of the virus (Waehler et al., 2007). Viral envelopes can be engineered in a variety of different ways to target desired tissues. Gene transfer using viral vectors can occur in vivo, with the vectors either applied locally or introduced systemically. However, viruses may also be applied to stem cells which are then reintroduced to the patient (see ‘cell-based delivery’ below; Waehler et al., 2007).
Electroporation is a method to introduce aqueous pores into cell membranes by exposing cells to pulsing electric fields (Yarmush, Golberg, Serša, Kotnik, & Miklavčič, 2014). Pores in the membrane bilayer typically remain open for milliseconds but may last for several minutes (Yarmush et al., 2014). These aqueous pores allow passage of genetic material through the membrane bilayer, transfecting the targeted tissues (Kalli, Teoh, & Leen, 2014). The electric fields required for electroporation can be produced by externally applied electrodes, and have been shown to cause no long-term side effects, though in the short term patients experience pain and muscle contractions. Another potential downside is that the transfection efficiency of electroporation varies depending on the type of tissue, and is generally lower than the transduction efficiency achieved by viral vectors (Yarmush et al., 2014).
Tissue particle bombardment
Another method of transfecting cells is tissue particle bombardment, also called the ‘gene gun’. Plasmids containing recombinant genes for transfection are coated onto inert gold particles 0.5-3µm in diameter, which are then accelerated to high speeds using either electrodes or pressurized helium (Kitagawa, Iwazawa, Robbins, Lotze, & Tahara, 2003; Yang, Burkholder, Roberts, Martinell, & McCabe, 1990). The particles are then fired at cells at high velocity, allowing them to penetrate the cell membranes and deliver the recombinant genes into the cytoplasm. The size, density and velocity of the particles can be adjusted to allow them to penetrate different types of tissue at different depths, ensuring that only the targeted cells will be transfected (Yang et al., 1990). The transfection efficiency of tissue particle bombardment is very low and usually leads to only transient expression (Kitagawa et al., 2003). This is because the genes carried by the gold particles are not incorporated by the genome but are instead expressed as episomes, which are degraded over time (Kitagawa et al., 2003).
One method of ensuring lasting, specific expression of a therapeutic agent is to use stem cells as a method of vector delivery (Sorrentino, 2002). In stem cell gene therapy, adult or embryonic stem cells are isolated in vitro and transfected or transduced with the genes of interest, then introduced into the patient. The stem cells used can be autologous (i.e. isolated from and reintroduced to the same patient), or allogeneic, (i.e. obtained from a different donor) (Wu & Dunbar, 2011). Engineering isolated cells in vitro is generally easier than attempting to transfect or transduce cells in vivo. Once introduced, all cell lineages that derive from the transgenic cells will carry the genes of interest. Because stem cells are capable of proliferating indefinitely, the transgenic cells will continue to deliver the gene therapy for the remainder of the patient’s life (Sorrentino, 2002). This makes it possible to permanently correct disease phenotypes; however, the permanent nature of the stem cells also poses significant risks. If the transgenic stem cells acquire unwanted mutations, they can proliferate unsustainably and form tumours (Wu & Dunbar, 2011). Once reintroduced to the patient, engineered stem cells could be very difficult to eliminate in case of harmful side effects. However, these risks can be mitigated by implementing improved protocols for the engineering and screening of stem cells in vitro (Wu & Dunbar, 2011). Rapid advancements in the field of induced pluripotent stem cells and tissue-specific stem cells are making it increasingly possible to engineer stem cells in vitro effectively and without causing unwanted side effects.
GENE THERAPY TO TARGET CANCER
Cancer is a difficult disease to treat for several reasons: malignant cells live amongst non-cancerous and often essential tissues; they can metastasize to relocate to other parts of the body; and most forms of cancer have some degree of resistance towards current treatments (Hanahan & Weinberg, 2011) In order to overcome these challenges, a therapy must be chosen which allows for the selective targeting of cancer cells over healthy cells. Ideally, this method could be administered systemically rather than locally, so as to counter metastases in addition to the primary tumour. In addition, it is important to use therapies to which cancer cells cannot easily develop a high degree of resistance. Notably, malignant cells exhibit a range of genetic and phenotypic abnormalities which can allow them to be selectively targeted using the gene therapy toolkit (McCormick, 2001). Several gene therapy techniques aimed at destroying cancer cells are under development. The next section of this review will draw upon the techniques described in the “Tools for Gene Therapy” section to outline potential combinations of therapeutic agent and delivery methods that have been successful or may be successful in the future. These methods can be grouped generally into two main categories: introducing genes toxic to cancer cells, such as tumour suppressor genes; and shutting off oncogene expression in malignant tissues, as shown in Figure 2.
Killing Tumor Cells
One method of killing cancer cells without harming healthy tissues is to use viral vectors, liposomes, or hydrogels to selectively deliver a toxic gene. Exciting results have been achieved using viral vectors to exploit different vulnerabilities in cancer cells. For instance, Gendicine, the first gene therapy in the world to be approved for clinical use, is an adenoviral vector which delivers a recombinant tumour suppressor gene (Chen et al., 2014; Pearson, Jia, & Kandachi, 2004). Inactivation of the tumour suppressor, p53, is a critical step in the development of many types of cancers (Chen et al., 2014; Lang et al., 2003). Therefore, application of Gendicine can be used to reactivate p53 and induce programmed cell death in cancer cells (Chen et al., 2014). Additionally, researchers found that Shigatoxin1A1 adenovirus vectors could be used to deliver gene products that are trans-spliced together (using SmaRT technology) inside of cells to encode a gene with a promoter activated only in cancerous cells (Nakayama, Pergolizzi, & Crystal, 2005). The trans-spliced mRNA encoded a toxin which induced apoptosis in cancer cells (Nakayama et al., 2005). Since the viral vector delivered two DNA fragments coding for 5′ and 3′ fragments of pre-mRNA of Shigatoxin1A1 instead of one DNA fragment coding for the entire gene, vectors could be grown in cells without the toxin killing the hosts (Nakayama et al., 2005).
Liposomes have fewer side effects than viruses, but are also less stable. Suzuki et al (2010) used liposomes to deliver the immunotherapeutic cytokine gene Interleukin-12 (IL-12) to tumour sites. The transgene was introduced systemically within ‘bubble liposomes’, which can be collapsed by ultrasound. Tumour cells were sonicated to collapse the liposomes in the area surrounding the malignant cells, ensuring that the therapeutic gene was introduced to the tumour specifically. The treatment caused cancerous tissue to experience inhibited protein production and inhibited growth (Suzuki et al., 2010). This is an example of a ‘remote triggering system’ which allows liposomes to release their contents only when near the tumour; other methods include light-sensitive and magnetically-responsive liposomes (Suzuki et al., 2010). However, liposomes normally have low efficacy because they degrade easily and often release their contents before reaching the tumour cells (Mufamadi et al., 2011).
New gene therapy techniques have also been applied to the challenge of treating cancer and have produced promising results. For example, hydrogels have been used to deliver therapeutic genes. In order to treat bone cancer, a thermal and pH sensitive chitosan hydrogel has been reported to successfully treat mice by delivering the small molecule Doxorubicin (Ta, Dass, Larson, Choong, & Dunstan, 2009). Doxorubicin can be toxic to healthy cells as well as malignant ones, but the advantage of the hydrogel was that different formulations could be composed to tailor the release of this chemical at cancerous tissues (Ta et al., 2009). In future trials, hydrogels could represent an ideal replacement for viral delivery because of their low toxicity (Seidlits et al., 2014). One attractive proposal would be to incorporate liposomes inside a hydrogel. This formulation has been shown to allow a controlled, yet stable drug release (Mufamadi et al., 2011).
Lastly, gene therapy can be used in conjunction with either chemotherapy or radiotherapy to improve their efficacy. An example of this is delivery of genetically modified hematopoietic stem cells (HSCs). HSCs in the bone marrow are often killed off by chemotherapy, causing severe side effects for the patient (Sorrentino, 2002). By introducing genetically modified, chemotherapy-resistant HSCs, the dose of chemotherapy can safely be increased. A recent study used this method to target glioblastomas, cancers which are often resistant to chemotherapy (Adair et al., 2012). Three patients were treated with autologous hematopoietic stem cells transduced with a retroviral vector to overexpress the gene P140K, which confers resistance to the chemotherapeutic drug O6-benzylguanine (6BG). The number of hematopoietic stem cells in all three patients dropped after each cycle of chemotherapy; however, each decrease was followed by an increase in the stem cell count, demonstrating that the population of hematopoietic stem cells was able to recover. An increase in P140K-modified cells was observed following each cycle of chemotherapy, suggesting that the introduction of P140K was responsible for conferring chemoprotection, demonstrating the potential of this combinatorial approach (Adair et al., 2012).
Targeting Oncogene Expression
Another important aim in developing cancer therapy is to knock out mutant oncogenes by repressing their expression or removing them from the genome. Gene therapy treatments for correcting genetic diseases offer interesting insight.
Several of the gene therapy tools designed to alter harmful genes have only been tested in animal models but have significant therapeutic potential. The first demonstration of CRISPR as a therapeutic strategy in vivo corrected a diseased phenotype in a mouse model of hereditary tyrosinemia, a disease caused by error of metabolism (Yin et al., 2014). A gene coding for the Cas9 nuclease, a single guide RNA, and a donor oligonucleotide were injected into the tail of the mouse, resulting in reconstitution of the wild type gene in mouse hepatocytes. Only about 0.4% of cells were corrected, a result which was likely due to low transfection efficiencies (Yin et al., 2014). Preclinical trials involving other methods of genome editing have also been successful. A novel combination of techniques was recently used to tackle recessive dystrophic epidermolysis bullosa, a disease caused by a single point mutation to the COL7A1 gene on chromosome three (Osborn et al., 2013). This point mutation usually results in a lack of type VII collagen protein, which leads to fatal skin blistering. Osborn et al. (2013) showed that co-delivery of TALEN DNA and an oligonucleotide donor to patient fibroblasts was able to rescue type VII collagen production. Further, the gene-edited fibroblasts were induced to return to the pluripotent state and then injected into mice, where they went on to produce skin-like structures as healthy, differentiated cells would (Osborn et al., 2013).
The new genome editing methods using TALENs and the CRISPR/Cas9 system show promise in pre-clinical trials when combined with viral vectors for delivery. Adenoviral vectors can carry a large enough amount of genetic material (‘genetic payload’) to introduce DNA encoding for TALENS or CRISPR oligonucleotides into the target cells, allowing cells to be transduced with much greater efficiency. A recent study has shown that adenoviral vectors can be used to transduce a range of human cells in vitro with genes coding for Cas9 nuclease and single guide RNA molecules, resulting in genome modifications at the targeted loci (Maggio et al., 2014). These modifications were achieved with an efficiency of 18-65 percent, depending on the tissue type. Adenoviral vectors have also been used to successfully achieve gene transfer of TALENs into human cells in vitro (Holkers et al., 2013)
Gene therapy has incredible potential for treating cancer because it can be used to target cancer cells on the basis of genetic defects rather than rapid proliferation, making treatments much more specific to tumour cells. This means that gene therapy could be used to treat cancer without the drastically harmful side effects of chemotherapy and radiotherapy, and can destroy metastases and micro-tumours which cannot be removed surgically. However, emerging gene therapies must overcome significant challenges before they can be used to treat cancer patients on a large scale.
Early gene therapy trials such as the SCID-XI trial in 2000 have called into question the safety of gene therapy, particularly the use of retroviral vectors (Wu & Dunbar, 2011). The failure of the SCID-XI trial showed that retroviral vectors can cause off-target mutations in the host cells, leading to uncontrolled replication and tumour formation. This is a concern for some applications of gene therapy to treat cancer, particularly the use of drug-resistant stem cells to mitigate the harmful effects of chemotherapy: if the modified stem cells start proliferating at an uncontrolled rate, the patient will experience the formation of a new, drug-resistant tumour (Wu & Dunbar, 2011). The study by Adair et al. (2012) successfully used retroviral vectors to transduce hematopoietic stem cells and confer a drug-resistance gene. To reduce the risk of mutagenesis, retroviral vectors could be replaced by adenoviral vectors carrying a Cas9 nuclease and gRNA. This approach would combine the high transduction efficiency of viral vectors with the specificity of CRISPR, thereby preventing unwanted mutations which could lead to harmful results.
Another risk of viral vectors is that genetically engineered viruses can trigger an immune response in the patient that destroys the therapeutic vectors. Tait et. al. (1999) showed that viral vectors can be used to destroy tumours, but only if the vectors can evade the immune system long enough to infect the cancer cells. Therefore, viral vectors can be a useful tool for treating immunocompromised patients such as those who have undergone many rounds of chemotherapy. Patients with healthy immune systems may benefit more from alternate methods of gene delivery, such as liposomes, tissue particle bombardment or electroporation. These methods can be used to deliver vectors without triggering an immune response, but are much less efficient than viral vectors. One promising method of increasing the efficiency of liposomes is to combine hydrogels and liposomes to make the vectors more stable. Similarly, hydrogels could potentially be used in conjunction with electroporation to ensure that the vectors transfect as many cells as possible. Electroporation and tissue particle bombardment are most useful for local application of gene therapy, whereas liposomes could be used for systemic application.
Previous experiments with liposomes, electroporation and tissue particle bombardment have mainly been conducted with these delivery methods transferring DNA constructs which are expressed as episomes (Mufamadi et al., 2011; Yang et al., 1990; Yarmush et al., 2014). Therefore, these trials have encountered the issue of transiency: once transfected, the host cells only express the transgene for a short amount of time. This can be a problem if, for example, the aim is to permanently shut down expression of an oncogene. One solution to this problem could be to combine these delivery methods with CRISPR or TALENs, which edit the genomic DNA of the host cells and therefore can induce long-lasting gene expression or knockdown. Yin et al. (2014) demonstrated that while CRISPR can be used to correct gene expression, simply injecting the Cas9 nuclease, guide RNA, and donor oligonucleotide did not result in a high enough transfection efficiency to treat disease in humans. Delivering the necessary components with liposomes, however, could increase the transfection efficiency enough to effectively correct disease phenotypes. The combination of delivery methods such as liposomes and hydrogels with therapeutic agents such as CRISPR and TALENs has the potential to be a powerful tool for gene therapy. Alternatively, combining technologies such as CRISPR with viral delivery systems could result in a highly specific and efficient method for genome editing.
Gene therapy is a relatively new but very promising solution to one of the most important challenges in the field of biotechnology today: the treatment of cancer. Using the gene therapy toolbox outlined in this review, it is possible to specifically target cancer cells by delivering toxic genes to tumour cells, or by altering the expression of oncogenes. Many of the methods in the toolbox have been demonstrated in clinical or preclinical trials to be specific, effective and long lasting inside the complex system of the body. These trials have shown that these methods already have a great deal of control over the types of genes that can be modified and how those genes can be modified. However, technologies such as CRISPR and SmaRT must still be put to the test in clinical trials. There are many barriers still to be overcome before gene therapy can become successful and easily applied to disease. With time, perhaps gene therapy can become a healthier alternative or complement to the radiotherapy and chemotherapy that is currently the primary cancer fighting tactic.
Adair, J. E., Beard, B. C., Trobridge, G. D., Neff, T., Rockhill, J. K., Silbergeld, D. L., Mrugala, Maciej M., Kiem, H.-P. (2012). Extended survival of glioblastoma patients after chemoprotective HSC gene therapy. Science Translational Medicine, 4(133), 133ra57.
Allen, T. M., & Cullis, P. R. (2013). Liposomal drug delivery systems: from concept to clinical applications. Advanced Drug Delivery Reviews, 65(1), 36–48.
American Cancer Society. (2016). Cancer facts & figures 2016.
Braun, C. J., Boztug, K., Paruzynski, A., Witzel, M., Schwarzer, A., Rothe, M., Modlich, U., Beier, R., Göhring, G., Steinemann, D., Fronza, R., Ball, C. R., Haemmerle, R., Naundorf, S., Kühlcke, K., Rose, M., Fraser, C., Mathias, L., Ferrari, R., Abboud, M. R. Al-Herz, W., Kondratenko, I., Maródi, L., Glimm, H., Schlegelberger, B., Schambach, A., Albert, M. H., Schmidt, M., von Kalle, C., Klein, C. (2014). Gene therapy for Wiskott-Aldrich syndrome--long-term efficacy and genotoxicity. Science Translational Medicine, 6(227), 227ra33.
Caló, E., & Khutoryanskiy, V. V. (2014). Biomedical applications of hydrogels: a review of patents and commercial products. European Polymer Journal, 65, 252–267.
Cartier, N., Hacein-Bey-Abina, S., Bartholomae, C. C., Bougnères, P., Schmidt, M., Kalle, C. V., Fischer, A., Cavazzana-Calvo, M., Aubourg, P. (2012). Lentiviral hematopoietic cell gene therapy for X-linked adrenoleukodystrophy. Methods in Enzymology, 507, 187–98.
Chen, G.X., Zhang, S., He, X.H., Liu, S.Y., Ma, C., & Zou, X.P. (2014). Clinical utility of recombinant adenoviral human p53 gene therapy: current perspectives. OncoTargets and Therapy, 7, 1901–9.
Dow, L. E., Fisher, J., O’Rourke, K. P., Muley, A., Kastenhuber, E. R., Livshits, G., Tschaharganeh, D. F., Socci, N. D., Lowe, S. W. (2015). Inducible in vivo genome editing with CRISPR-Cas9. Nature Biotechnology, 33(4), 390–394.
Gaj, T., Gersbach, C. A., & Barbas, C. F. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology, 31(7), 397–405.
Gori, J. L., Hsu, P. D., Maeder, M. L., Shen, S., Welstead, G. G., & Bumcrot, D. (2015). Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy. Human Gene Therapy, 26(7), 443–51.
Hacein-Bey-Abina, S., Garrigue, A., Wang, G. P., Soulier, J., Lim, A., Morillon, E., Clappier, E., Caccavelli, L., Delabesse, E., Beldjord, K., Asnafi, V., MacIntyre, E., Dal Cortivo, L., Radford, I., Brousse, N., Sigaux, F., Moshous, D., Hauer, J., Borkhardt, A., Belohradsky, B. H., Wintergerst, U., Velez, M. C., Leiva, L., Sorensen, R., Wulffraat, N., Blanche, S., Bushman, F. D., Fischer, A., Cavazzana-Calvo, M. (2008). Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. The Journal of Clinical Investigation, 118(9), 3132–42.
Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646–74.
Hockemeyer, D., Wang, H., Kiani, S., Lai, C. S., Gao, Q., Cassady, J. P., Cost, G. J., Zhang, L., Santiago, Y., Miller, J. C., Zeitler, B., Cherone, J. M., Meng, X., Hinkley, S. J., Rebar, E. J., Gregory, P. D., Urnov, F. D., Jaenisch, R. (2011). Genetic engineering of human pluripotent cells using TALE nucleases. Nature Biotechnology, 29(8), 731–4.
Holkers, M., Maggio, I., Liu, J., Janssen, J. M., Miselli, F., Mussolino, C., Recchia, A., Cathomen, T., Gonçalves, M. A. F. V. (2013). Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells. Nucleic Acids Research, 41(5), e63.
Kaliberov, S. A., & Buchsbaum, D. J. (2012). Chapter seven--Cancer treatment with gene therapy and radiation therapy. Advances in Cancer Research, 115, 221–63.
Kalli, C., Teoh, W. C., & Leen, E. (2014). Introduction of Genes via Sonoporation and Electroporation. Advances in Experimental Medicine and Biology, 818, 231–54.
Kitagawa, T., Iwazawa, T., Robbins, P. D., Lotze, M. T., & Tahara, H. (2003). Advantages and limitations of particle-mediated transfection (gene gun) in cancer immuno-gene therapy using IL-10, IL-12 or B7-1 in murine tumor models. The Journal of Gene Medicine, 5(11), 958–65.
Kootstra, N. A., & Verma, I. M. (2003). Gene therapy with viral vectors. Annual Review of Pharmacology and Toxicology, 43, 413–39.
Lang, F., Bruner, J., Fuller, G., Aldape, K., Prados, M., Chang, S., Berger, M., McDermott, M., Kunwar, S., Junck, L., Chandler, W., Zwiebel, J., Kaplan, R., Yung, A. (2003). Phase I Trial of Adenovirus-Mediated p53 Gene Therapy for Recurrent Glioma: Biological and Clinical Results. Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology, 21(13), 2508–2518.
Maggio, I., Holkers, M., Liu, J., Janssen, J. M., Chen, X., & Gonçalves, M. A. F. V. (2014). Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Scientific Reports, 4, 5105.
Malik, P., & Arumugam, P. I. (2005). Gene Therapy for beta-thalassemia. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program, 45–50.
Marraffini, L. A., & Sontheimer, E. J. (2010). CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nature Reviews. Genetics, 11(3), 181–90.
McCormack, M. P., Young, L. F., Vasudevan, S., de Graaf, C. A., Codrington, R., Rabbitts, T. H., Jane, S., Curtis, D. J. (2010). The Lmo2 oncogene initiates leukemia in mice by inducing thymocyte self-renewal. Science (New York, N.Y.), 327(5967), 879–83.
McCormick, F. (2001). Cancer gene therapy: fringe or cutting edge? Nature Reviews. Cancer, 1(2), 130–41.
Mufamadi, M. S., Pillay, V., Choonara, Y. E., Du Toit, L. C., Modi, G., Naidoo, D., & Ndesendo, V. M. K. (2011). A review on composite liposomal technologies for specialized drug delivery. Journal of Drug Delivery, 2011, 939851.
Nakayama, K., Pergolizzi, R. G., & Crystal, R. G. (2005). Gene transfer-mediated pre-mRNA segmental trans-splicing as a strategy to deliver intracellular toxins for cancer therapy. Cancer Research, 65(1), 254–63.
Osborn, M. J., Starker, C. G., McElroy, A. N., Webber, B. R., Riddle, M. J., Xia, L., DeFeo, A. P., Gabriel, R., Schmidt, M., von Kalle, C., Carlson, D. F., Maeder, M. L., Joung, J. K., Wagner, J. E., Voytas, D.F., Blazar, B. R., Tolar, J. (2013). TALEN-based gene correction for epidermolysis bullosa. Molecular Therapy : The Journal of the American Society of Gene Therapy, 21(6), 1151–9.
Pearson, S., Jia, H., & Kandachi, K. (2004). China approves first gene therapy. Nature Biotechnology, 22(1), 3–4.
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA-Guided Platform for Sequence- Specific Control of Gene Expression, 152(5), 1173–1183.
Seidlits, S. K., Gower, R. M., Shepard, J. A., & Shea, L. D. (2014). Hydrogels for Lentiviral Gene Delivery. Expert Opinions on Drug Delivery, 10(4), 499–509.
Siegel, R. L., Miller, K. D., & Jemal, A. (2015). Cancer statistics, 2015. CA: A Cancer Journal for Clinicians, 65(1), 5–29.
Sorrentino, B. P. (2002). Gene therapy to protect haematopoietic cells from cytotoxic cancer drugs. Nature Reviews. Cancer, 2(6), 431–41.
Suzuki, R., Namai, E., Oda, Y., Nishiie, N., Otake, S., Koshima, R., Hirata, K., Taira, Y., Utoguchi, N., Negishi, Y., Nakagawa, S., Maruyama, K. (2010). Cancer gene therapy by IL-12 gene delivery using liposomal bubbles and tumoral ultrasound exposure. Journal of Controlled Release : Official Journal of the Controlled Release Society, 142(2), 245–50.
Ta, H. T., Dass, C. R., Larson, I., Choong, P. F. M., & Dunstan, D. E. (2009). A chitosan-dipotassium orthophosphate hydrogel for the delivery of Doxorubicin in the treatment of osteosarcoma. Biomaterials, 30(21), 3605–13.
Tait, D. L., Obermiller, P. S., Hatmaker, A. R., Redlin-frazier, S., & Holt, J. T. (1999). Ovarian Cancer BRCA1 Gene Therapy : Phase I and II Trial Differences in Immune Response and Vector Stability Ovarian Cancer BRCA1 Gene Therapy : Phase I and II Trial Differences in Immune Response and Vector Stability 1. Clinical Cancer Research, 5, 1708–1714.
Urruticoechea, A., Alemany, R., Balart, J., Villanueva, A., Viñals, F., & Capellá, G. (2010). Recent advances in cancer therapy: an overview. Current Pharmaceutical Design, 16(1), 3–10.
Waehler, R., Russell, S. J., & Curiel, D. T. (2007). Engineering targeted viral vectors for gene therapy. Nature Reviews. Genetics, 8(8), 573–87.
Wilkinson, R., & Wiedenheft, B. (2014). A CRISPR method for genome engineering. F1000prime Reports, 6, 3.
Wu, C., & Dunbar, C. E. (2011). Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity. Frontiers of Medicine, 5(4), 356–71.
Yang, N. S., Burkholder, J., Roberts, B., Martinell, B., & McCabe, D. (1990). In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proceedings of the National Academy of Sciences of the United States of America, 87(24), 9568–72.
Yang, Y., & Walsh, C. E. (2005). Spliceosome-mediated RNA trans-splicing. Molecular Therapy : The Journal of the American Society of Gene Therapy, 12(6), 1006–12.
Yarmush, M. L., Golberg, A., Serša, G., Kotnik, T., & Miklavčič, D. (2014). Electroporation-Based Technologies for Medicine: Principles, Applications, and Challenges. Annual Review of Biomedical Engineering, 16, 295–320.
Yin, H., Xue, W., Chen, S., Bogorad, R. L., Benedetti, E., Grompe, M., Koteliansky, V., Sharp, P. A., Jacks, T., Anderson, D. G. (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnology, 32(6), 551–3.