**4. Targeting in non-viral systems**

Early approaches to gene therapy involved direct introduction of genetic material into locally accessible tumours. While this proved useful in some cases, the invasive nature of these methods renders them impractical for internal and disseminated tumours. Therefore, a systemically administered cancer gene therapy vector that can target tumours is ideal. Traditional chemotherapeutic cytotoxic drugs cause such harsh and debilitating side effects because they affect rapidly dividing cells and do not differentiate between normal or cancerous cells. In order to avoid these off-target effects, it is necessary to target the therapeutic directly to the cancer cells without affecting normal healthy cells. Improved knowledge of cellular, metabolic, and signalling pathways essential for tumour growth has led to the identification of targets on cancer cells [91]. Different types of cancerous cells tend to have distinct charac‐ teristics, which set them apart from normal cells, meaning that a range of potential molecular targets exists that can be targeted to direct gene therapy towards tumours. Cancer cells typically overexpress certain proteins on their surface, which may be exploited through targeting strategies; commonly overexpressed proteins include integrins [92], transferrin receptors [46], epidermal growth factor receptors (EGFR) [93], folate receptors [94], and proteoglycans [95], and targeting such receptors has been found to increase the specificity and efficacy of drug delivery, while reducing side effects [96]. Active targeting using ligands that target overexpressed receptors specific to cancer cells is therefore an attractive targeting strategy for systemically administered non-viral gene delivery and much research has focused around this.

#### **4.1. Targeting ligands**

Through phage display techniques, ligands for specific receptors commonly overexpressed on cancer cells have been successfully identified and incorporated into vectors [97]. When the delivery vector reaches the tumour environment, the overexpressed receptors bind the ligand on the surface of the vector and it is subsequently internalised via receptor-mediated endocy‐ tosis. This strategy serves to both target the cells and facilitate uptake by cells, but the endocytic pathway used can depend on the targeting ligand and cell type [98]. RGD peptide is a commonly used peptide targeting ligand. It is a tripeptide of Arg-Gly-Asp that was derived from fibronectin, which mediates cell attachment. RGD peptide is involved in cell adhesion to cell surface integrins [99]. Integrin receptors, such as αvβ3 integrin and related αv-integrins, are commonly upregulated on the surface of angiogenic endothelial cells and can have a profound effect on the ability of tumours to survive and progress through regulation of stemness, metastasis, and resistance [100]. This activity makes integrins valuable for targeting aggressive cancers and various strategies have been used to incorporate RGD into non-viral delivery systems for targeting angiogenic tumours [101]. Kim et al. presented a tumourtargeting, RGD-conjugated, bio-reducible polymer for the delivery of vascular endothelial growth factor (VEGF) siRNA. The RGD-functionalised vectors showed 20–59% higher cellular uptake in MCF-7 breast cancer cells and PANC-1 pancreatic cancer cells compared to nontargeted vectors. In addition, MCF-7 and PANC-1 cancer cells had significantly reduced VEGF gene expression (51–71%) and cancer cell viability (35–43%) compared with control [102]. Nie et al. described a dual functionalised system that boasts two targeting ligands, namely RGD and B6 peptide, which target transferrin receptors. Transfection efficiency of the dual targeting system resulted in 8- and 4-fold higher luciferase reporter gene expression compared to single targeted control formulations with either B6 or RGD in DU145 and PC3 prostate cancer cells, respectively [103].

When formulating a targeted non-viral gene delivery system, there are a number of factors to be considered that may have an effect on the overall efficiency of the delivery system, such as ligand density and positioning on the surface of the vector, vector size, and choice of targeting ligand [90]. Vector ligand density should be optimised to ensure efficient binding to receptors. Furthermore, the binding of a ligand to its substrate may facilitate binding of neighbouring receptors in a thermodynamically favourable way [104]. In addition, the strategies used to link the targeting moieties to vectors, as well as many other factors including relative specificity, expression of target receptors, and physiological factors need to be considered in order to improve effectiveness and avoid interference or loss of biological activity [105].

One such problem with active targeting is that it is dependent on the expression of certain receptors by tumour cells. In breast cancer, oestrogen, progesterone, and human epidermal growth factor (HER) receptors have been identified and targeted. However, 15% of breast cancers, termed triple negative, are defined by a lack of these receptors. Absence of these receptors makes such cancers difficult to target and treat, hence patients with triple-negative disease have poorer prognoses [106]. Therefore, there is a need for a more general targeting strategy that targets the common characteristics of cancer cells and is not reliant on the expression of any one receptor. This would also broaden the scope of disease states that may be treated using any individual gene therapy strategy, making them more marketable for the pharmaceutical industry. Additionally, drug resistance can develop if mutation of cancer cells affects the expression of the target receptors. The receptors may be down-regulated resulting in reduced targetability and subsequent reduced cellular uptake of the vectors, or conversely up-regulation of receptors could render the vector inefficient. Receptors may also be expressed in different isoforms, altering their recognition of the targeting moiety [107]. Moreover, heterogeneity of tumours may result in different levels of receptor expression within a single tumour [108].

Although ligand-targeted vectors have proven to be safe and efficacious in preclinical models, it has not yet been unambiguously proven that targeting ligands contribute to the efficacy of vectors, and it seems that targeting ligands do not cause localisation within the target tissue, but rather provide benefits in terms of internalisation to target cells and retention at the target site once the delivery system has arrived [109]. While this method of targeting may enhance non-viral gene delivery systems, it has not completely met expectations and other targeting strategies have been explored.

#### **4.2. Targeting at the transcriptional level**

**4. Targeting in non-viral systems**

70 Gene Therapy - Principles and Challenges

around this.

**4.1. Targeting ligands**

Early approaches to gene therapy involved direct introduction of genetic material into locally accessible tumours. While this proved useful in some cases, the invasive nature of these methods renders them impractical for internal and disseminated tumours. Therefore, a systemically administered cancer gene therapy vector that can target tumours is ideal. Traditional chemotherapeutic cytotoxic drugs cause such harsh and debilitating side effects because they affect rapidly dividing cells and do not differentiate between normal or cancerous cells. In order to avoid these off-target effects, it is necessary to target the therapeutic directly to the cancer cells without affecting normal healthy cells. Improved knowledge of cellular, metabolic, and signalling pathways essential for tumour growth has led to the identification of targets on cancer cells [91]. Different types of cancerous cells tend to have distinct charac‐ teristics, which set them apart from normal cells, meaning that a range of potential molecular targets exists that can be targeted to direct gene therapy towards tumours. Cancer cells typically overexpress certain proteins on their surface, which may be exploited through targeting strategies; commonly overexpressed proteins include integrins [92], transferrin receptors [46], epidermal growth factor receptors (EGFR) [93], folate receptors [94], and proteoglycans [95], and targeting such receptors has been found to increase the specificity and efficacy of drug delivery, while reducing side effects [96]. Active targeting using ligands that target overexpressed receptors specific to cancer cells is therefore an attractive targeting strategy for systemically administered non-viral gene delivery and much research has focused

Through phage display techniques, ligands for specific receptors commonly overexpressed on cancer cells have been successfully identified and incorporated into vectors [97]. When the delivery vector reaches the tumour environment, the overexpressed receptors bind the ligand on the surface of the vector and it is subsequently internalised via receptor-mediated endocy‐ tosis. This strategy serves to both target the cells and facilitate uptake by cells, but the endocytic pathway used can depend on the targeting ligand and cell type [98]. RGD peptide is a commonly used peptide targeting ligand. It is a tripeptide of Arg-Gly-Asp that was derived from fibronectin, which mediates cell attachment. RGD peptide is involved in cell adhesion to cell surface integrins [99]. Integrin receptors, such as αvβ3 integrin and related αv-integrins, are commonly upregulated on the surface of angiogenic endothelial cells and can have a profound effect on the ability of tumours to survive and progress through regulation of stemness, metastasis, and resistance [100]. This activity makes integrins valuable for targeting aggressive cancers and various strategies have been used to incorporate RGD into non-viral delivery systems for targeting angiogenic tumours [101]. Kim et al. presented a tumourtargeting, RGD-conjugated, bio-reducible polymer for the delivery of vascular endothelial growth factor (VEGF) siRNA. The RGD-functionalised vectors showed 20–59% higher cellular uptake in MCF-7 breast cancer cells and PANC-1 pancreatic cancer cells compared to nontargeted vectors. In addition, MCF-7 and PANC-1 cancer cells had significantly reduced VEGF gene expression (51–71%) and cancer cell viability (35–43%) compared with control [102]. Nie

In an attempt to overcome these problems with the targeting of proteins expressed by cancer cells, it has been suggested that targeting the upstream genetic causes of dysregulated genes may be more successful [110]. Regulation of gene expression at the transcriptional level for cancer gene therapy can occur in a cell-specific manner with a focus on tissue-specific and tumour-specific promoters, or alternatively the use of inducible promoters, which allow gene expression to be controlled exogenously by factors such as heat or radiation. The major drawback with tissue-specific promoters, however, is that toxic gene expression occurs in all cells in the tissue, both healthy and cancerous, which limits use of this method to tissues that are not critical to the survival of the patient such as thyroid or prostate [111]. Use of inducible promoters to drive transgene expression requires the activation by exogenous factors, but the tumour specificity that this strategy affords may be useful in supplementing the potency of other therapies, such as the use of a radiation-inducible promoter for enhancement of iNOS transgene expression [112]; this dual approach to therapy can limit toxic effects in normal cells. However, for simplicity, we will focus solely on tumour-specific promoters.

The complex interplay of various factors involved in gene expression is often altered in cancer cells, and through exploiting this genetic signature of cancer, reduced off target effects and toxicity should result. Certain genes are upregulated in cancer through the over activation of transcription factors, which activate the upstream promoter of these genes. This can then be exploited to give tumour-specific targeting by using promoters that are activated by tran‐ scription factors known to be overexpressed in cancer cells to drive expression of the transgene in tumour cells only. Tumour-specific promoters are sub-categorized as follows: cancer specific promoters, tumour-type specific promoters, tumour microenvironment-related promoters, and tumour vasculature-related promoters, and are extensively reviewed by [113] and [114].

#### *4.2.1. Cancer-specific promoters*

The identification of genes that are expressed in cancer cells only may lead to targeting of cancer gene therapy in a broad sense regardless of cancer type. One such example is telomerase, involved in telomere maintenance, which is considered crucial in the progression and immortalisation of cancer cells and is expressed in the vast majority of cancers [115]. Telomer‐ ase expression is regulated by the human telomerase reverse transcriptase subunit promoter (hTERTp), which was recently used by Xie et al. in a non-viral delivery system. The hTERTp promoter was used to drive expression of a transgene amplification vector VISA (VP16-GAL4- WPRE integrated systemic amplifier) to target a phosphoprotein that is enriched in astrocytes (PEA-15) in advanced breast tumours. PEA-15 is known to affect signal-regulated kinase (ERK) in the cytoplasm, thereby inhibiting cell proliferation and inducing apoptosis [116]. Transgene expression was found to be highly specific, inducing cancer-cell killing in breast cancer cell lines (T47D, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-361, MDA-MB-453, BT474, 4T1, SKBR-3) in vitro without affecting normal mammary epithelial cells (184A1 and MCF-10A). Furthermore, an in vivo study in a MDA-MB-231 xenograft mouse model demonstrated that the expression of PEA-15 driven by the hTERTp driven VISA vector prolonged mouse survival more effectively than PEA-15 driven by cytomegalovirus (CMV) promoter whilst showing no acute toxicities. The authors demonstrated that the use of the hTERT promoter achieved targeting and selective cell kill in triple negative MDA-MB-231 breast cancer cells, a selectivity that was lacking when transgene expression was promoted by the CMV promoter [116].

Survivin is a protein that functions in the inhibition of apoptosis, therefore, its overexpression in cancer cells can facilitate uninhibited growth. It is known to be upregulated in cancer cells and expression is controlled by various transcription factors including nuclear factor kappa B (NF-kB), Runx2 and the Ras family that bind to the survivin promoter, triggering expression [117, 118]. The survivin promoter (pSURV) has therefore been incorporated into gene delivery systems to drive transgene expression preferentially in cancer cells. Qu et al. used pSURV to drive the expression of the herpes simplex virus thymidine kinase (HSVtk) gene for suicide gene therapy. The authors used pSURV/GFP to demonstrate that gene expression occurred in HepG2 hepatocellular carcinoma cells, while no gene expression was observed in LO2 normal human liver cells. Apoptotic rates of up to 55% were achieved in HepG2 cells with pSURV/ HSVtk demonstrating the possibility of this system for suicide gene therapy. However, further in vivo studies need to be carried out to properly assess the targeting ability of this system [119].

#### *4.2.2. Tumour-type specific promoters*

cancer gene therapy can occur in a cell-specific manner with a focus on tissue-specific and tumour-specific promoters, or alternatively the use of inducible promoters, which allow gene expression to be controlled exogenously by factors such as heat or radiation. The major drawback with tissue-specific promoters, however, is that toxic gene expression occurs in all cells in the tissue, both healthy and cancerous, which limits use of this method to tissues that are not critical to the survival of the patient such as thyroid or prostate [111]. Use of inducible promoters to drive transgene expression requires the activation by exogenous factors, but the tumour specificity that this strategy affords may be useful in supplementing the potency of other therapies, such as the use of a radiation-inducible promoter for enhancement of iNOS transgene expression [112]; this dual approach to therapy can limit toxic effects in normal cells.

The complex interplay of various factors involved in gene expression is often altered in cancer cells, and through exploiting this genetic signature of cancer, reduced off target effects and toxicity should result. Certain genes are upregulated in cancer through the over activation of transcription factors, which activate the upstream promoter of these genes. This can then be exploited to give tumour-specific targeting by using promoters that are activated by tran‐ scription factors known to be overexpressed in cancer cells to drive expression of the transgene in tumour cells only. Tumour-specific promoters are sub-categorized as follows: cancer specific promoters, tumour-type specific promoters, tumour microenvironment-related promoters, and tumour vasculature-related promoters, and are extensively reviewed by [113] and [114].

The identification of genes that are expressed in cancer cells only may lead to targeting of cancer gene therapy in a broad sense regardless of cancer type. One such example is telomerase, involved in telomere maintenance, which is considered crucial in the progression and immortalisation of cancer cells and is expressed in the vast majority of cancers [115]. Telomer‐ ase expression is regulated by the human telomerase reverse transcriptase subunit promoter (hTERTp), which was recently used by Xie et al. in a non-viral delivery system. The hTERTp promoter was used to drive expression of a transgene amplification vector VISA (VP16-GAL4- WPRE integrated systemic amplifier) to target a phosphoprotein that is enriched in astrocytes (PEA-15) in advanced breast tumours. PEA-15 is known to affect signal-regulated kinase (ERK) in the cytoplasm, thereby inhibiting cell proliferation and inducing apoptosis [116]. Transgene expression was found to be highly specific, inducing cancer-cell killing in breast cancer cell lines (T47D, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-361, MDA-MB-453, BT474, 4T1, SKBR-3) in vitro without affecting normal mammary epithelial cells (184A1 and MCF-10A). Furthermore, an in vivo study in a MDA-MB-231 xenograft mouse model demonstrated that the expression of PEA-15 driven by the hTERTp driven VISA vector prolonged mouse survival more effectively than PEA-15 driven by cytomegalovirus (CMV) promoter whilst showing no acute toxicities. The authors demonstrated that the use of the hTERT promoter achieved targeting and selective cell kill in triple negative MDA-MB-231 breast cancer cells, a selectivity that was lacking when transgene expression was promoted by the CMV promoter [116].

However, for simplicity, we will focus solely on tumour-specific promoters.

*4.2.1. Cancer-specific promoters*

72 Gene Therapy - Principles and Challenges

Many different types of cancer overexpress various genes, which are characteristic of that tumour type, and the promoters responsible for this expression can then be exploited for tumour-type specific targeting. Osteocalcin is a protein normally found in the bone matrix but has been found to be elevated in cancers such as ovarian and prostate cancer and is associated with the progression and formation of bone metastases; McCarthy's group used the human osteocalcin promoter (hOC) for tumour-limited gene expression. It has been shown that hOC has strong promoter activity in cancer cells, with transcription factors such as Runx2 involved in gene upregulation [120, 121]. In this case, hOC was used to drive the expression of inducible nitric oxide synthase expression in PC-3 and DU145 prostate cancer cells. The authors demonstrated significant delay in tumour growth with no toxic side effects in vivo*,* highlight‐ ing the potential for hOC to target prostate cancer tumours [122]. The advantage of using this tumour-type specific promoter is that it may facilitate the targeting of the primary tumour, as well as disseminated metastatic lesions that are often the most aggressive and hardest to treat forms of cancer. Figure 2 represents the targeting strategy of a tumour-type specific promoter that is activated in cancerous cells but not in normal cells.

Figure 2 summarises active internalisation of gene delivery vector and initiation of transgene expression in a non-transformed and a transformed cell. Gene delivery vectors are commonly functionalised using an antibody that targets HER-2 [123], while the human osteocalcin promoter has been employed to drive inducible nitric oxide synthase gene expression in prostate and breast cancer cells [122].

#### *4.2.3. Tumour microenvironment-related promoters*

The tumour microenvironment provides a unique environment that provides ideal conditions for growth and progression of tumours. Hypoxic conditions are often associated with chemoand radio-resistance in tumours, and hypoxia is thought to be a key element for the cancer stem cell niche [124]. Various genes have been identified to be upregulated in the hypoxic environment with hypoxia response elements (HREs) working in concert with transcription factors, such as HIF-1, to activate transcription in response to hypoxia. Fujioka et al. reported

the construction of a vector combining a hypoxia response promoter with the CMV promoter (HRE-CMV) that resulted in a 2-fold increase in apoptotic gene expression compared to expression driven by CMV alone. In vivo, BCL-2 shRNA activity driven by the HRE-CMV promoter in hypoxic colon 26 tumours resulted in tumour volume reduction that was signif‐ icantly greater than when bcl-2 shRNA was driven by CMV alone [125]. Although this study demonstrates the action of the HRE promoter for treatment in hypoxic tumours, the authors used intra-tumoural injections to deliver the vector, which does not give an indication of the tumour targeting specificity of this strategy.

#### *4.2.4. Tumour vasculature-related promoters*

The ability of tumours to trigger angiogenesis for increased tumour blood supply has been associated with more aggressive tumours, metastases, and poor prognosis. Identification of the genes involved in this process has led to the use of promoters that can be exploited for targeting. One such example is VEGF, which has been shown to have a major role in tumour angiogenesis by activating tyrosine kinase receptors VEGFR1 (Flt-1) and VEGFR2 (kinase insert receptor (KDR) in humans/Flk-1 in mice). KDR was found to be overexpressed in activated endothelial cells of newly formed vessels and strongly associated with invasion and metastasis in human malignant diseases [126]. Wang et al used the KDR promoter to drive thymidine kinase (TK) gene expression, which activated the prodrug ganciclovir (GCV) for suicide gene therapy. The authors demonstrated that the KDR promoter and TK/GCV showed a targeted killing effect on transfected human umbilical vein endothelial cells (HUVEC). Cells transfected with KDR-TK were 2- to 5-fold more sensitive to GCV compared to non-transfected HUVEC and HepG2 cells [127]. Again, however, confirmation of these impressive in vitro results in an in vivo setting using systemic delivery is required to validate tumour targetability and efficacy of the suicide gene/prodrug system.
