Emma M. McErlean, Cian M. McCrudden and Helen O. McCarthy

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61297

#### **Abstract**

This chapter examines key concepts with respect to cancer gene therapy and the cur‐ rent issues with respect to non-viral delivery. The biological and molecular barriers that need to be overcome before effective non-viral delivery systems can be appropri‐ ately designed for oncology applications are highlighted and ways to overcome these are discussed. Strategies developed to evade the immune response are also described and targeted gene delivery is examined with the most effective strategies highlighted. Finally, this chapter proposes a new way forward based on a growing body of evi‐ dence that supports a multifunctional delivery approach involving the creation of vec‐ tors, with a unique molecular architecture designed using a bottom-up approach.

**Keywords:** Cancer gene therapy, Multifunctional delivery systems, Non-viral gene delivery, Bio-inspired vectors, Tumour-targeted delivery

#### **1. Introduction**

Progress in the treatment of cancer in recent times has been unprecedented as cancer survival in the UK has doubled in the last 40 years, with 50% of adult cancer patients diagnosed in 2010– 2011 in England and Wales predicted to survive 10 years or more [1]. Improvements in cancer screening techniques have led to improved prognosis through early detection, with breast cancer screening estimated to prevent up to 1,300 deaths per year; women who are diagnosed with the earliest stage of breast cancer have a 90% 5-year survival rate [2]. Likewise, if diagnosed early, prostate cancer responds well to treatments such as hormone therapy, with 65–90% of men diagnosed in stage 1 or 2 likely to live at least 10 years post diagnosis [3]. Despite improvements in screening and early detection methods, conventional treatment options are not always effective. Mainstay cancer treatment options including radiotherapy, chemother‐

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apy, and surgery are extremely arduous on patients, and often have only moderate success. Existing anticancer drugs are generally cytotoxic, but lack specificity for the target tumour, which results in severe side effects. Not only do these traditional therapies cause damage to healthy cells, they are rarely effective against all transformed cells, and sometimes lack potency entirely. Failure of treatment can result in disease recurrence, often in a more aggressive form, with chemo- and radio-resistant aggressive malignancies ensuing. Resistance to conventional therapy causes treatment failure in over 90% of patients with advanced metastatic cancer [4].

Gene therapy is an exciting research area that involves the delivery of genetic material into cells to alter their function. Diseases that arise consequential of anomalous DNA (e.g., cystic fibrosis [5, 6]) are appropriate for gene therapy intervention; as cancer's origins lie in DNA damage, this group of diseases is also particularly suitable for gene-based therapeutics [7]. The strategy of gene therapy is generally to replace or repair faulty genes by the transfer and insertion of corrective or therapeutic genes [8]. Alternatively, the strategy of gene therapy can be the supraphysiological expression of cytotoxic proteins, or the expression of proteins to affect metabolism of prodrugs, for direct or indirect cytocidal effects, respectively [9]. Silencing of problematic genes is a nascent and very popular strategy that uses RNAi therapeutics to inhibit the expression of certain undesirable genes at the post-transcriptional level [10]. Some of the most common strategies of cancer gene therapy include suicide gene therapy, tumour suppressor gene therapy, antiangiogenic therapy, and cancer immunotherapy [11].

#### **1.1. Tumour suppressor gene therapy**

Mutation of tumour suppressor genes, such as p53, has been highlighted as a mechanism of proliferation and resistance in some cancers. Functional p53 interacts with other cellular pathways including the death-receptor pathways and caspases, as well as inhibiting antiapoptotic mediators, such as the BCL family, leading to the suppression of tumour growth [12]. For this reason, the delivery of transgenes that encode tumour suppressor genes, including genes encoding p53, IL-2, EGFR, and E1A, has received considerable attention. Promising results have been obtained, with Senzer et al. reporting the systemic administration of targeted liposomal nanoparticles with an anti-transferrin receptor targeting moiety (TfR); they deliv‐ ered the p53 tumour suppressor gene in complexes known as SGT-53 for advanced solid tumours in a phase I clinical trial [13]. SGT-53 was administered to patients with a range of advanced cancer types, including cervical cancer, thyroid cancer, and colorectal cancer. Median survival was 340 days and 7 of the 11 patients treated exhibited stable disease at 6 week assessment, with one patient reclassified from inoperable to operable due to significant tumour necrosis. The authors also demonstrated the tumour targeting ability of SGT-53 with biopsies of tumour tissue and normal tissue; normal tissue showing negligible exogenous p53 levels. However, the main aim of the study was to assess safety and further studies would be required to fully assess therapeutic effects.

#### **1.2. Suicide gene therapy**

Suicide gene therapy, also known as gene-directed enzyme-producing therapy (GDEPT), involves the administration of an enzyme-encoding transgene to the tissue, with a separate administration of a relatively innocuous prodrug. Transgene expression produces the enzyme within cancerous cells and subsequently the prodrug is converted to its toxic form by this enzyme [14]. GDEPT strategies include cytosine deaminase/F-fluorocytosine (CD/5-FC), where the CD transgene metabolises 5-FC to 5-Fluorouracil (5-FU); herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) where HSVtk coverts GCV to its cytotoxic triphosphate derivative; and *E. coli* nitroreductase/CB1954 (NTR/CB) where NTR activates the prodrug CB1954 resulting in toxicity to tumour cells [15]. Multiple GDEPT systems have made it to clinical trials and Sangro et al. reported on a recent phase 1 clinical trial of HSVtk/GCV in the treatment of advanced hepatocellular carcinoma. Intra-tumoural injection of the TK gene in a replication deficient adenovirus vector (Ad-TK) was followed with systemic administra‐ tion of GCV in 10 patients [16]. Although the main aims of the trial were to assess feasibility and safety of treatment, anti-tumour effects were also assessed. Stabilisation of tumour was observed in 60% of patients, with two patients who received the high dose treatment showing tumour necrosis and one patient surviving for 26 months. Such therapies, which may be used in addition to radiotherapy, have the advantage of being activated only in the cancerous cells due to direct intra-tumoural delivery, reducing toxic side-effects to normal cells. A 'bystander effect' where neighbouring cells receive the toxic treatment through gap junctions has also been observed, which could be beneficial for therapy as reduced amounts of treatment are needed for the same therapeutic effect; conversely, this bystander effect may limit the potential of the therapy if neighbouring healthy cells receive the toxic treatment. However, problems in vector development still need to be overcome before these treatments are to be successful [17]. Despite progression to clinical trials, no GDEPT therapy has made it to the market and the use of such treatment strategies may be limited to locally available tumour sites due to the need for intra-tumoural injection.

#### **1.3. Anti-angiogenic therapy**

apy, and surgery are extremely arduous on patients, and often have only moderate success. Existing anticancer drugs are generally cytotoxic, but lack specificity for the target tumour, which results in severe side effects. Not only do these traditional therapies cause damage to healthy cells, they are rarely effective against all transformed cells, and sometimes lack potency entirely. Failure of treatment can result in disease recurrence, often in a more aggressive form, with chemo- and radio-resistant aggressive malignancies ensuing. Resistance to conventional therapy causes treatment failure in over 90% of patients with advanced metastatic cancer [4].

Gene therapy is an exciting research area that involves the delivery of genetic material into cells to alter their function. Diseases that arise consequential of anomalous DNA (e.g., cystic fibrosis [5, 6]) are appropriate for gene therapy intervention; as cancer's origins lie in DNA damage, this group of diseases is also particularly suitable for gene-based therapeutics [7]. The strategy of gene therapy is generally to replace or repair faulty genes by the transfer and insertion of corrective or therapeutic genes [8]. Alternatively, the strategy of gene therapy can be the supraphysiological expression of cytotoxic proteins, or the expression of proteins to affect metabolism of prodrugs, for direct or indirect cytocidal effects, respectively [9]. Silencing of problematic genes is a nascent and very popular strategy that uses RNAi therapeutics to inhibit the expression of certain undesirable genes at the post-transcriptional level [10]. Some of the most common strategies of cancer gene therapy include suicide gene therapy, tumour

suppressor gene therapy, antiangiogenic therapy, and cancer immunotherapy [11].

Mutation of tumour suppressor genes, such as p53, has been highlighted as a mechanism of proliferation and resistance in some cancers. Functional p53 interacts with other cellular pathways including the death-receptor pathways and caspases, as well as inhibiting antiapoptotic mediators, such as the BCL family, leading to the suppression of tumour growth [12]. For this reason, the delivery of transgenes that encode tumour suppressor genes, including genes encoding p53, IL-2, EGFR, and E1A, has received considerable attention. Promising results have been obtained, with Senzer et al. reporting the systemic administration of targeted liposomal nanoparticles with an anti-transferrin receptor targeting moiety (TfR); they deliv‐ ered the p53 tumour suppressor gene in complexes known as SGT-53 for advanced solid tumours in a phase I clinical trial [13]. SGT-53 was administered to patients with a range of advanced cancer types, including cervical cancer, thyroid cancer, and colorectal cancer. Median survival was 340 days and 7 of the 11 patients treated exhibited stable disease at 6 week assessment, with one patient reclassified from inoperable to operable due to significant tumour necrosis. The authors also demonstrated the tumour targeting ability of SGT-53 with biopsies of tumour tissue and normal tissue; normal tissue showing negligible exogenous p53 levels. However, the main aim of the study was to assess safety and further studies would be

Suicide gene therapy, also known as gene-directed enzyme-producing therapy (GDEPT), involves the administration of an enzyme-encoding transgene to the tissue, with a separate

**1.1. Tumour suppressor gene therapy**

58 Gene Therapy - Principles and Challenges

required to fully assess therapeutic effects.

**1.2. Suicide gene therapy**

In contrast to suicide gene therapy and tumour suppressor therapy, which are quite specific in focus, targeting angiogenesis may attack the root of the greater tumour establishment. There are various 'classical' protein-based angiogenesis inhibitors, including receptor tyrosine kinase inhibitors, which block the activity of vascular endothelial growth factor (VEGF), and mono‐ clonal antibodies against VEGF-A such as bevacizumab (Avastin®). However, as angiogenesis is required for normal function in the body, such as wound healing, complete blockade of angiogenesis is not desirable. In addition, it may seem that rather than kill tumours, inhibitor therapy may merely retard further tumour growth. Moreover, the existence of various resistance mechanisms to angiogenic inhibitors, including alternative signalling pathways poses major drawbacks to such therapy. Alternatively, a gene therapy approach targeting the genes behind the pro-angiogenic factors may be more suitable [18, 19]. Doan et al. described a gene silencing approach that halts the effects of the pro-angiogenic factors VEGF and kinesin spindle protein (KSP), which play a critical role in cellular proliferation [20]. Hep3B hepato‐ cellular carcinoma cells were treated with a cocktail of anti-VEGF and anti-KSP siRNAs, and a significant reduction in both VEGF and KSP expression was observed; in vitro, this mani‐ fested reduced proliferation of the cells, assessed by WST-1 assay and clonogenic survival assay. The results demonstrate the potential for anti-angiogenic gene therapy, but translation to in vivo studies is required to establish this further.

#### **1.4. Cancer immunotherapy**

Cancer immunotherapy is the process of harnessing the immune system to attack cancer cells. Cancer cells present antigens, known as tumour-associated antigens (TAAs) or tumourspecific antigens (TSAs) on their surface; recognition of these antigens by immune cells has been exploited in the development of cancer DNA vaccines. Cancer vaccines may be prophy‐ lactic or therapeutic in their design, which would generate an active immune response specifically to tumours while also providing memory cells to control future recurrence [21]. DNA encoding the genes for TAAs is delivered to cancer cells that subsequently express the transgenic TAA, eliciting an immune response against the tumour cells and many cancer DNA vaccines are being assessed in clinical trials [22]. Chudley et al. reported on a phase I/II clinical trial of a DNA vaccine encoding a domain (DOM) from fragment C of a tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA) in patients with prostate cancer [23]. Following intramuscular administration of the DNA vaccine to 30 patients, 29 had a measurable CD4+ T-cell response and PSMA-specific CD8+ T cells were detected in 16/30 patients. As a result, PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months denoting slower progression of the disease. Staff et al. reported on a phase I clinical trial in patients with colorectal cancer with a DNA vaccine [24]. The plasmid vaccine was administered by Biojector® and encoded human carcinoembryonic antigen (CEA), which is known to be over expressed by a large number of epithelial neoplasias, including colorectal cancer. No serious adverse effects were observed with the vaccine and of the 10 patients, 8 showed no evidence of disease at follow-up. However, despite the promising trial results and 4 DNA vaccines licensed for use in animals including Oncept® for Canine Melanoma [25], no product has made it to the market in humans. Many trials use direct injection of the vaccines to tumours and efficiency may be enhanced if delivery vectors were to be used, which could maximise transduction.

To date, cancer has been the most common disease focus for gene therapy, with 64% of all ongoing gene therapy clinical trials targeting a malignancy [26]. However, the progress of gene therapy beyond clinical trials has been disappointing, with only three products currently having made it to the market, namely, Gendicine®, Oncorine®, and Glybera®. Gendicine® and Oncorine®, which deliver p53 tumour suppressor genes for the treatment of head and neck cancer, are licenced in China; while Glybera®, used for the treatment of severe lipoprotein lipase deficiency, is the only gene therapy product licensed in Europe [27]. Despite the many promising therapeutic strategies for gene therapy, the common limiting factor has been the lack of a suitable delivery vehicle that has the ability to specifically target tumour cells, whilst being non-immunogenic and non-toxic. Consequently, a vast amount of research has therefore focused on delivery systems for gene therapy. In order for the potential of gene therapy to be realised, the focus needs to be on the design of an appropriate delivery vehicle that will meet all the demands in terms of functionality and satisfaction of regulatory bodies.

### **2. The biological barriers to gene delivery**

The safest way to deliver gene therapy is by direct administration of the therapeutic to the target site. However, this is extremely inefficient, unreliable, and feasible only in tumours in superficial sites. Generally, gene therapy approaches are delivered via the intravenous route; as nucleic acids are susceptible to degradation by nucleases and rapid clearance in systemic circulation [28], a vector is required to package, protect, and transport the genetic material to its site of action.

Viral vectors, derived from naturally evolved viruses capable of transferring their genetic material into host cells, remain the most efficient gene delivery agents [29]. However, difficulty in large scale production, limitation in size of DNA that can be carried, and concerns about mutagenesis, toxicity, and immunogenicity have hindered the progression of viral vectors [30, 31]. As a result, much research has focused on the design of non-viral vectors, which have the potential to circumvent the problems associated with viral vectors [32]. Non-viral gene delivery encompasses a wide variety of delivery systems including cationic polymers, liposomes, proteins, and peptides that have the ability to package nucleic acids and deliver them into cells [33]. However, transfection efficiency of non-viral vectors remains significantly lower than viral vectors [34], and many factors are to be considered and hurdles overcome when designing an efficient non-viral delivery system. Successful gene therapy relies largely on the development of an efficient vector that can overcome the various extracellular and intracellular barriers to deliver the genetic material to its target site [35].

#### **2.1. Extracellular barriers**

**1.4. Cancer immunotherapy**

60 Gene Therapy - Principles and Challenges

to be used, which could maximise transduction.

**2. The biological barriers to gene delivery**

Cancer immunotherapy is the process of harnessing the immune system to attack cancer cells. Cancer cells present antigens, known as tumour-associated antigens (TAAs) or tumourspecific antigens (TSAs) on their surface; recognition of these antigens by immune cells has been exploited in the development of cancer DNA vaccines. Cancer vaccines may be prophy‐ lactic or therapeutic in their design, which would generate an active immune response specifically to tumours while also providing memory cells to control future recurrence [21]. DNA encoding the genes for TAAs is delivered to cancer cells that subsequently express the transgenic TAA, eliciting an immune response against the tumour cells and many cancer DNA vaccines are being assessed in clinical trials [22]. Chudley et al. reported on a phase I/II clinical trial of a DNA vaccine encoding a domain (DOM) from fragment C of a tetanus toxin linked to an HLA-A2-binding epitope from prostate-specific membrane antigen (PSMA) in patients with prostate cancer [23]. Following intramuscular administration of the DNA vaccine to 30 patients, 29 had a measurable CD4+ T-cell response and PSMA-specific CD8+ T cells were detected in 16/30 patients. As a result, PSA doubling time increased significantly from 11.97 months pre-treatment to 16.82 months denoting slower progression of the disease. Staff et al. reported on a phase I clinical trial in patients with colorectal cancer with a DNA vaccine [24]. The plasmid vaccine was administered by Biojector® and encoded human carcinoembryonic antigen (CEA), which is known to be over expressed by a large number of epithelial neoplasias, including colorectal cancer. No serious adverse effects were observed with the vaccine and of the 10 patients, 8 showed no evidence of disease at follow-up. However, despite the promising trial results and 4 DNA vaccines licensed for use in animals including Oncept® for Canine Melanoma [25], no product has made it to the market in humans. Many trials use direct injection of the vaccines to tumours and efficiency may be enhanced if delivery vectors were

To date, cancer has been the most common disease focus for gene therapy, with 64% of all ongoing gene therapy clinical trials targeting a malignancy [26]. However, the progress of gene therapy beyond clinical trials has been disappointing, with only three products currently having made it to the market, namely, Gendicine®, Oncorine®, and Glybera®. Gendicine® and Oncorine®, which deliver p53 tumour suppressor genes for the treatment of head and neck cancer, are licenced in China; while Glybera®, used for the treatment of severe lipoprotein lipase deficiency, is the only gene therapy product licensed in Europe [27]. Despite the many promising therapeutic strategies for gene therapy, the common limiting factor has been the lack of a suitable delivery vehicle that has the ability to specifically target tumour cells, whilst being non-immunogenic and non-toxic. Consequently, a vast amount of research has therefore focused on delivery systems for gene therapy. In order for the potential of gene therapy to be realised, the focus needs to be on the design of an appropriate delivery vehicle that will meet

The safest way to deliver gene therapy is by direct administration of the therapeutic to the target site. However, this is extremely inefficient, unreliable, and feasible only in tumours in

all the demands in terms of functionality and satisfaction of regulatory bodies.

Although delivery vectors have the ability to protect the DNA from endonuclease attack, the vectors themselves may also be susceptible to recognition and clearance. In the systemic circulation, vectors may be rapidly cleared from circulation by the reticulo-endothelial system (RES), also known as the mononuclear phagocyte system (MPS) [36]. Many non-viral delivery vectors are cationic in nature, a desirable characteristic for condensing DNA and promoting cellular uptake. However, this cationic nature can be problematic for systemic administration due to interaction with blood components, such as serum proteins, which may result in opsonisation. Consequently, large aggregates are formed that cannot traverse cell membranes and may become lodged in microvascular networks or accumulate in MPS organs such as the liver or spleen [37]. Further to this, cationic systems may interact with cell membranes indiscriminately, affecting normal cells as well as cancerous cells, and strong cationic charges can induce damage of cellular membranes and apoptosis [38]. Thus, a balance must be reached in the design of a delivery vector such that the nucleic acid/vector complex has cationicity of appropriate magnitude, so as to permit proper association with target cells whilst preventing aggregation.

The circulation of gene therapy delivery systems is often cut short due to rapid hepatic metabolism and clearance, and often this clearance occurs before the particles can reach their target site to deliver the therapeutic. The use of 'stealth particles', such as those that contain polyethylene glycol (PEG), has been shown to increase the circulation time of various delivery systems by shielding the charge of the particles, reducing binding with serum proteins and aggregation, whilst evading the immune system [39]. Various strategies in evading this clearance have been employed, which will be discussed in a later section. However, if the vector can avoid clearance, extravasation from blood circulation needs to occur in order to reach the tumour cells, which can be hindered by the chaotic blood supply, poor permeability, and high interstitial pressure within the tumour [40].

#### **2.2. Intracellular barriers**

Surviving the systemic circulation and reaching the target cell is not the only hurdle faced by non-viral gene delivery systems. The nature of conventional gene therapy requires the genetic material to be transcribed by the cell, which requires delivery to the nucleus (or in the case of siRNA technology, delivery to the cytoplasm). A number of intracellular barriers exist that may impede this delivery include traversing the cell membrane, escape from the endosome, and release of the nucleic acid payload into the cytoplasm, followed by active transport to the nucleus with subsequent nuclear import.

#### *2.2.1. Cell membrane/internalisation*

Cell membranes are lipophilic anionic structures that are generally impermeable to large macromolecular anionic nucleic acids [32]. Non-viral gene delivery systems aim to complex nucleic acid cargo, thereby masking their native negative charge, to give an overall net cationic complex capable of interaction with cell membranes. Not only does this allow for electrostatic interactions between the vector and the membrane, it also condenses the DNA to a size suitable for cellular uptake (≤200 nm diameter). Various pathways of cellular uptake exist that are size dependent. For example, the cell penetrating peptide TAT, derived from the human immu‐ nodeficiency-1 virus (HIV-1) [41], enters cells via different routes depending on the size of the cargo. Larger cargoes of proteins or quantum dots that exceed the 500 Daltons restriction limit are internalised with TAT via the caveolae or macropinocytosis routes, and smaller cargoes such as peptides less than 30–40 amino acids via the clathrin route [42]. Further to this, the internalisation route may also depend on other factors such as cell type, receptors present on the cell, temperature, incubation time, concentration of the vector, and properties of the vector including cargo and linkage type [7, 43]. Different internalisation pathways also have an effect on the fate of the vector once inside the cell. As a result, much research has centred around elucidating the mechanisms involved in cellular uptake in order to improve the efficiency of gene therapy [44]. Endocytosis (clathrin-mediated, caveolae-mediated, or macropinocytosis) is thought to be the main uptake pathway for most gene delivery.

#### *2.2.2. Clathrin-Mediated Endocytosis (CME)*

CME is the most well-defined route of endocytosis and involves the internalisation of cargo via receptors on the cell membrane, such as proteoglycans, into vesicles known as clathrin coated pits, which are about 100–150 nm in diameter. These pits are transported via microtu‐ bules of the cell cytoskeleton deeper into the cell, where they form endosomes (acidic, degradative compartments that transport material back to the membrane for recycling, or to lysosomes for degradation). The term 'receptor-mediated endocytosis' is often used to describe CME, however, endocytosis via receptors does not exclusively occur via CME [45]. The addition of ligands, such as transferrin, to delivery systems has allowed for targeting to cancer cells overexpressing the transferrin receptor that binds to the ligand and facilitates internali‐ sation via CME [46].

#### *2.2.3. Caveolae-Mediated Endocytosis (CvME)*

CvME is initiated by flask-shaped invaginations known as caveolae that have lipid-raft formations involving cholesterol and sphingolipids, which are around 50–200 nm in diameter. Internalisation occurs in an actin-dependent manner, forming a type of endosome known as a caveosome. Caveosomes are not as acidic or destructive as CME endosomes, but can still ultimately merge with the lysosomal machinery [47]. It has been observed that many com‐ monly used cancer cells lines (e.g., PC-3 prostate cancer cells) lack the ability to form caveolae that may have significance for delivery systems relying on this route for internalisation [48]. Furthermore, it has been observed that caveolae may be upregulated in some cancer cells providing a possible target for delivery systems. Nguyen et al. reported that a polysorbitolmediated transporter (PSMT) was used to deliver plasmid DNA encoding the p53 tumour suppressor gene into human cervical cancer (HeLa) cells and normal human diploid fibroblast (HDF) cells. PSMT entered cancer cells selectively via CvME with transgene expression resulting in cellular damage and apoptosis [49].

#### *2.2.4. Macropinocytosis*

**2.2. Intracellular barriers**

62 Gene Therapy - Principles and Challenges

nucleus with subsequent nuclear import.

*2.2.1. Cell membrane/internalisation*

Surviving the systemic circulation and reaching the target cell is not the only hurdle faced by non-viral gene delivery systems. The nature of conventional gene therapy requires the genetic material to be transcribed by the cell, which requires delivery to the nucleus (or in the case of siRNA technology, delivery to the cytoplasm). A number of intracellular barriers exist that may impede this delivery include traversing the cell membrane, escape from the endosome, and release of the nucleic acid payload into the cytoplasm, followed by active transport to the

Cell membranes are lipophilic anionic structures that are generally impermeable to large macromolecular anionic nucleic acids [32]. Non-viral gene delivery systems aim to complex nucleic acid cargo, thereby masking their native negative charge, to give an overall net cationic complex capable of interaction with cell membranes. Not only does this allow for electrostatic interactions between the vector and the membrane, it also condenses the DNA to a size suitable for cellular uptake (≤200 nm diameter). Various pathways of cellular uptake exist that are size dependent. For example, the cell penetrating peptide TAT, derived from the human immu‐ nodeficiency-1 virus (HIV-1) [41], enters cells via different routes depending on the size of the cargo. Larger cargoes of proteins or quantum dots that exceed the 500 Daltons restriction limit are internalised with TAT via the caveolae or macropinocytosis routes, and smaller cargoes such as peptides less than 30–40 amino acids via the clathrin route [42]. Further to this, the internalisation route may also depend on other factors such as cell type, receptors present on the cell, temperature, incubation time, concentration of the vector, and properties of the vector including cargo and linkage type [7, 43]. Different internalisation pathways also have an effect on the fate of the vector once inside the cell. As a result, much research has centred around elucidating the mechanisms involved in cellular uptake in order to improve the efficiency of gene therapy [44]. Endocytosis (clathrin-mediated, caveolae-mediated, or macropinocytosis)

CME is the most well-defined route of endocytosis and involves the internalisation of cargo via receptors on the cell membrane, such as proteoglycans, into vesicles known as clathrin coated pits, which are about 100–150 nm in diameter. These pits are transported via microtu‐ bules of the cell cytoskeleton deeper into the cell, where they form endosomes (acidic, degradative compartments that transport material back to the membrane for recycling, or to lysosomes for degradation). The term 'receptor-mediated endocytosis' is often used to describe CME, however, endocytosis via receptors does not exclusively occur via CME [45]. The addition of ligands, such as transferrin, to delivery systems has allowed for targeting to cancer cells overexpressing the transferrin receptor that binds to the ligand and facilitates internali‐

is thought to be the main uptake pathway for most gene delivery.

*2.2.2. Clathrin-Mediated Endocytosis (CME)*

sation via CME [46].

Macropinocytosis involves the uptake of large amounts of fluid-phase materials. It occurs via an actin-driven mechanism that causes ruffling of the cell membrane to form protrusions that engulf the extracellular material into macropinosomes, which eventually merge with the endosomal pathway [50]. Anaka et al. reported that macropinocytosis was the main cellular uptake pathway of the peptide STR-CH2R4H2C when complexed with plasmid DNA and delivered to COS7 kidney fibroblast cells, attributing the position of arginine residues exposed on the surface of the complexes as the reason for this internalisation route [51].

#### *2.2.5. Direct internalisation*

Cationic vectors, especially those rich in arginine, have been observed to enter cells via nonendocytic routes through direct internalisation triggered by non-specific electrostatic interac‐ tions [52]. This form of internalisation is a more attractive route for non-viral gene delivery, as direct delivery into the cytoplasm avoids the endosome. In the case of arginine-rich cell penetrating peptides, an initial electrostatic interaction with the cell membrane is followed by formation of a peptide-cargo-phospholipid complex with the positively charged guanidium group of arginine bound to the phosphate groups of the phosphatidylcholine (PC) and/or sphingomyelin (SM) of the outer leaflet of the cell membrane. A 'capacitor' is then formed between the cationic arginine residues and the anionic phosphatidylserine creating an electric field strong enough to form a reversible pore, which allows the CPP-cargo to pass through the membrane [53]. Arginine-rich peptides, such as octa-arginine (R8), have therefore been utilised for gene delivery due their strong cell penetrating ability. However, little is known about this entry route, and evidence suggests that vectors can enter cells via multiple mechanisms [54]. Understanding the various routes through which vectors can enter cells can aid the gene therapist in the design of vectors; ensuring appropriate size and charge, for example, can allow for targeted internalisation via a specific mechanism.

#### **2.3. Endosomal entrapment**

Following endocytosis, vectors may be trapped in the endosomal pathway. Endosomal entrapment poses a major limiting step to efficient gene therapy. The endosomal compartment provides cells with a way of regulating what enters and leaves the cell and material within the endosome can be either recycled to the cell membrane or progressed to lysosomes. It is essential that therapeutics escape the endosome in order to avoid degradation of the nucleic acid payload [55]. Endosomal escape can be achieved by different mechanisms and typically nonviral delivery systems are designed to facilitate this. The 'proton sponge' effect is exploited by polymers that contain amine groups such as polyethylenimine (PEI) or by histidine-rich peptides, which have been used in many delivery systems for gene therapy [56]. Fusogenic peptides evoke membrane destabilisation by interacting with anionic lipids in the endosomal membrane, thereby disrupting the membrane, allowing release of the endosomal contents [57]. INF-7 peptide is an example of a synthetic fusogenic peptide derived from influenza virus hemagglutinin protein, which enhances endosomal escape. Oliviera et al. report that the addition of INF-7 peptide to Lipofectamine for delivery of anti-kRas siRNA resulted in 3.5 fold improved gene silencing effect and subsequent reduction in kRas protein expression in C26 murine colon carcinoma cells in vitro when compared to Lipofectamine/siRNA complexes alone [58].

#### **2.4. Intracellular trafficking**

Following endosomal escape, the vector and its cargo must be delivered to the correct cellular compartment, i.e., DNA delivered to the nucleus, or siRNA assembly into RNA-induced silencing complexes (RISC) in the cytoplasm [59, 60]. However, this is not without its challeng‐ es due to the restricted movement of macromolecules in the cell, slowing the mobility of vectors towards the nucleus [61], while endonucleases may degrade any naked nucleic acid [62]. The vector is therefore required to protect and transport the nucleic acid through the cytoplasm in order to reach its target organelle. Movement in the cytoplasm is restricted due to overcrowd‐ ingoforganelles,the cell cytoskeletonandhighproteinconcentrations,whichcollectivelyresult in a major impediment to non-viral delivery of even relatively small cargoes [63]. A network of microtubules and associated motor proteins (dyneins and kinesins) are responsible for the maintenance of correct organelle location [64] and intracellular transport of vesicles, lyso‐ somes, and endosomes [65]. If non-viral gene delivery vectors could utilise the microtubule networkwithinthecell,itwouldserveasadirectroutetothenucleusandtransfectionefficiencies may be greatly improved. Toledo et al. presented a recombinant fusion protein based on the dynein light chain LC8 that facilitated plasmid DNA uptake into HeLa cells and transported DNA via microtubules to the nucleus for GFP transgene expression [66].

#### **2.5. Nuclear import**

In the case of DNA gene therapy, once the vector reaches the nucleus, it must gain entry and deliver its genetic payload in order for the gene to be transcribed and elicit its effect. The nucleus is protected by a bilayer known as the nuclear envelope, and entry into the nucleus through the nuclear envelope is tightly controlled by the nuclear pore complex (NPC) [67]. The NPC only allows the passive entry of molecules that do not exceed 10 nm in diameter, which limits the entry of DNA; active traversing of the nuclear envelope is hence required. The NPC therefore poses the last major hurdle to gene therapy and is a huge rate-limiting step in transfection efficiency. The addition of short amino acid sequences known as nuclear localisation signals (NLS) to vectors has been useful in trafficking and facilitating nuclear entry [68]. The nuclear localisation signal from the simian virus 40 (SV40), large tumour antigen has been used to enhance transfection efficiency in many delivery systems. Wang et al. demon‐ strated that the addition of SV40 NLS to R8 resulted in a transfection efficiency of up to 80% as effective as jetPEITM (transfection reagent) with no cytotoxic effects in HeLa cells [69].

**2.3. Endosomal entrapment**

64 Gene Therapy - Principles and Challenges

alone [58].

**2.4. Intracellular trafficking**

**2.5. Nuclear import**

Following endocytosis, vectors may be trapped in the endosomal pathway. Endosomal entrapment poses a major limiting step to efficient gene therapy. The endosomal compartment provides cells with a way of regulating what enters and leaves the cell and material within the endosome can be either recycled to the cell membrane or progressed to lysosomes. It is essential that therapeutics escape the endosome in order to avoid degradation of the nucleic acid payload [55]. Endosomal escape can be achieved by different mechanisms and typically nonviral delivery systems are designed to facilitate this. The 'proton sponge' effect is exploited by polymers that contain amine groups such as polyethylenimine (PEI) or by histidine-rich peptides, which have been used in many delivery systems for gene therapy [56]. Fusogenic peptides evoke membrane destabilisation by interacting with anionic lipids in the endosomal membrane, thereby disrupting the membrane, allowing release of the endosomal contents [57]. INF-7 peptide is an example of a synthetic fusogenic peptide derived from influenza virus hemagglutinin protein, which enhances endosomal escape. Oliviera et al. report that the addition of INF-7 peptide to Lipofectamine for delivery of anti-kRas siRNA resulted in 3.5 fold improved gene silencing effect and subsequent reduction in kRas protein expression in C26 murine colon carcinoma cells in vitro when compared to Lipofectamine/siRNA complexes

Following endosomal escape, the vector and its cargo must be delivered to the correct cellular compartment, i.e., DNA delivered to the nucleus, or siRNA assembly into RNA-induced silencing complexes (RISC) in the cytoplasm [59, 60]. However, this is not without its challeng‐ es due to the restricted movement of macromolecules in the cell, slowing the mobility of vectors towards the nucleus [61], while endonucleases may degrade any naked nucleic acid [62]. The vector is therefore required to protect and transport the nucleic acid through the cytoplasm in order to reach its target organelle. Movement in the cytoplasm is restricted due to overcrowd‐ ingoforganelles,the cell cytoskeletonandhighproteinconcentrations,whichcollectivelyresult in a major impediment to non-viral delivery of even relatively small cargoes [63]. A network of microtubules and associated motor proteins (dyneins and kinesins) are responsible for the maintenance of correct organelle location [64] and intracellular transport of vesicles, lyso‐ somes, and endosomes [65]. If non-viral gene delivery vectors could utilise the microtubule networkwithinthecell,itwouldserveasadirectroutetothenucleusandtransfectionefficiencies may be greatly improved. Toledo et al. presented a recombinant fusion protein based on the dynein light chain LC8 that facilitated plasmid DNA uptake into HeLa cells and transported

In the case of DNA gene therapy, once the vector reaches the nucleus, it must gain entry and deliver its genetic payload in order for the gene to be transcribed and elicit its effect. The nucleus is protected by a bilayer known as the nuclear envelope, and entry into the nucleus through the nuclear envelope is tightly controlled by the nuclear pore complex (NPC) [67].

DNA via microtubules to the nucleus for GFP transgene expression [66].

Understanding the various barriers to gene delivery allows the rational design of delivery systems that can overcome these hurdles. The ideal non-viral gene delivery vector is a multifunctional system with the ability to condense DNA effectively, overcome the various intraand extracellular barriers and must also be non-toxic and non-immunogenic. Furthermore, vectors can be designed specifically to exploit the characteristics of cancer cells and tumours, including the enhanced permeability and retention (EPR) effect associated with tumour vasculature; where gene therapy delivery systems exploit the permeability of the tumour vasculature to localise and accumulate in the tumour through passive diffusion [70]. Other factors, including tumour microenvironment, and the aberrant expression of certain enzymes and proteins commonly associated with cancer cells may also be targeted or exploited. Figure 1 represents how a multi-functional non-viral gene delivery system may be composed.

**Figure 1.** Schematic of a multi-functionalised vector for therapeutic transgene delivery.

Anionic plasmid DNA cargo is condensed using a cationic material such as poly-L-lysine or protamine. Vectors are functionalised with adjuncts to aid in evasion of the various barriers that are posed to gene therapy strategies, as highlighted above. The various functional groups will be discussed below.
