**6. The future: Molecular engineering**

incorporating into RISC, where its guide-strand binds to and cleaves the complementary mRNA with a perfect match. The cleaved mRNA is subsequently released and the siRNA guide-strand-bound RISC is free to bind to another mRNA and start a new round of cleavage [132]. However, the short half-life of siRNA has resulted in production of shRNA, which has been developed as an alternative RNA molecule. Transcription of shRNA occurs in the nucleus from an expression vector that bears a short double-stranded DNA sequence with a hairpin loop. This shRNA transcript is then processed by RNase enzymes and incorporated into RISC

The use of siRNA and shRNA to silence unfavourable genes that are overexpressed in cancer has gained much attention. Multidrug resistance (MDR) genes, responsible for resistance to chemotherapeutics have been problematic in the treatment of cancer and associated with poor prognosis. By silencing these genes using siRNA, it has been possible to improve response to conventional treatments. For example, Chen et al. used siRNA to silence the MDR1 gene in doxorubicin resistant MCF-7 breast cancer cells, which resulted in 85–90% reduction in MDR1 gene expression and subsequently sensitisation of 70% of cells to doxorubicin [134]. Another approach is to target and silence pro-angiogenic genes such as the Notch pathway. Yang et al. used a non-viral delivery system to deliver siRNA for silencing the Notch-1 gene in breast cancer and found that transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis [135]. One advantage of using siRNA to down-regulate overexpressed proteins is that non-specific delivery is often less toxic than the delivery of plasmid DNA that encodes genes such as IL-2 and TNF-alpha. However, to limit any toxicity that does exist, many groups have added

targeting ligands to the delivery systems to increase tumour specificity [136].

MicroRNAs (miRNA) are highly conserved short non-coding RNAs that negatively regulate a wide range of physiological processes at the post-transcriptional level including apoptosis, proliferation, and migration [137]. Initially, miRNA is transcribed in the nucleus as a primary transcript (pri-miRNA), which is processed to give a two-nucleotide overhang at its 3' and is termed a pre-miRNA. Pre-miRNA is subsequently exported to the cytoplasm where it is further cleaved and mature miRNA is loaded into RISC to elicit its effect [138]. miRNAs can be either oncogenic or tumour suppressive in nature and as a result, may be overexpressed (e.g., miR-132, miR-20, and miR-17-92 family) or underexpressed (e.g., miR-34a and miR-126) in cancer cells making them targets for cancer gene therapy. A vast amount of information has been obtained in recent years on many different miRNAs and their role in cancer and with cancer stem cells, and by characterising their function, it may be possible to exploit them in

A single miRNA may have several varied targets to which it could bind and bring about gene silencing. miRNA-34a, known to be down-regulated in various cancers, has been shown to be a potent tumour suppressor that has various targets including the Notch pathway, BCL-2, survivin, c-Myc, and c-Met transcription factors [141]. Hu et al. demonstrated the value of miR-34a-mediated tumour suppression with the in vivo systemic administration of a non-viral

in the cytoplasm [133].

76 Gene Therapy - Principles and Challenges

**5.2. MicroRNA**

cancer gene therapy [139, 140].

Strategies involving PEGylation and the use of targeting ligands have shown great promise for cancer gene therapy in overcoming certain hurdles, but in order to maximise the efficiency of non-viral delivery, vectors must have the ability to overcome all the barriers to gene delivery. Recent research in the field has focused on the development of vectors for nucleic acid delivery that efficiently evade the barriers to gene delivery highlighted above, and provoke adequate transgene expression in vivo following systemic delivery.

#### **6.1. Multifunctional Envelope-type Nano Devices (MENDs)**

Harashima et al. presented a multifunctional envelope-type nano device (MEND) that was produced on the concept of 'programmed packaging' with a rational design to overcome barriers to delivery and assembly into nano-sized vectors. Generally, a MEND comprises a DNA core condensed using a cationic polymer such as poly-L-lysine (PLL), which is wrapped in a separate lipid envelope fortified with various functional attachments including targeting ligands, PEG, and groups facilitating cellular uptake and endosomal escape [144]. One of the first MEND systems described consisted of a PLL DNA condensing core, surrounded by a lipid envelope, and functionalised with stearylated octaarginine (R8) to promote cellular uptake to deliver anti-luciferase siRNA [145]. The gene silencing effect of the MEND was found to be comparable to that of the transfection reagent Lipofectamine 2000, without any detectable cytotoxicity and further optimisation of this system to include protamine as the DNA con‐ densing agent resulted in a 70% silencing effect in transfected COS7 fibroblast cells.

The nature of the MEND system renders it relatively easy to modify in order to optimise transfection efficiency. When the lipid component of egg phosphatidylcholine (EPC) and cholesterol were replaced with the fusogenic lipids DOPE and cholesteryl hemisuccinate (CHEMS), respectively, an overall 2-fold improvement was observed due to the optimisation of the lipid component [146]. Furthermore, functionalization of MEND with octa-arginine R8 (R8-MEND) for enhanced cellular uptake resulted in a transfection efficiency of more than 80% in HeLa cells [147]. The addition of pH-sensitive endosomal escape motifs to MEND, such as INF7 derived from the HA2 protein of the influenza virus envelope, has also proven beneficial, and the combination of INF7 with R8 resulted in the production of R8/INF7/MEND. In vivo administration of R8/INF7/MEND to ICF mice produced luciferase expression 240-fold higher in liver and 115-fold higher in spleen than that of R8-MEND alone, demonstrating the importance of optimising functionality of the MEND system [148].

In a similar approach, the pH-sensitive fusogenic peptide GALA was incorporated into a MEND system as an endosomal escape enhancer in a system that comprised of R8 and an MMP-cleavable PEG functionality. Increased gene silencing effect was observed for delivery of anti-luciferase siRNA in HeLa-luc cells in vitro when compared to an unmodified MEND [149]. In addition to this, an in vivo study using a HT1080-luc xenografted model demonstrated that the cleavable PEGylated GALA/R8/MEND exhibited efficient luciferase gene knockdown in comparison to PEG-MEND, which was unable to cause any gene knockdown.

Incorporation of targeting motifs has also proved useful in MEND systems. The addition of RGD peptide to MEND, which is a targeting ligand for integrins, resulted in significant tumour growth delay in OS-RC-2 human renal carcinoma bearing mice when RGD-MEND was used to deliver anti-VEGF siRNA in vivo [150]. GALA peptide was also used as a targeting ligand for sialic acid-terminated sugar chains on pulmonary endothelium as reported by Kusumoto et al. [151]. Following intravenous administration of GALA-MEND delivering antiCD31 siRNA in vivo, approximately 50% inhibition of lung metastasis in a Murine melanoma B16- F10 mouse model was observed when compared with control groups. Examples of MENDs and the various functionalities that have been employed are detailed in Table 1.



cytotoxicity and further optimisation of this system to include protamine as the DNA con‐

The nature of the MEND system renders it relatively easy to modify in order to optimise transfection efficiency. When the lipid component of egg phosphatidylcholine (EPC) and cholesterol were replaced with the fusogenic lipids DOPE and cholesteryl hemisuccinate (CHEMS), respectively, an overall 2-fold improvement was observed due to the optimisation of the lipid component [146]. Furthermore, functionalization of MEND with octa-arginine R8 (R8-MEND) for enhanced cellular uptake resulted in a transfection efficiency of more than 80% in HeLa cells [147]. The addition of pH-sensitive endosomal escape motifs to MEND, such as INF7 derived from the HA2 protein of the influenza virus envelope, has also proven beneficial, and the combination of INF7 with R8 resulted in the production of R8/INF7/MEND. In vivo administration of R8/INF7/MEND to ICF mice produced luciferase expression 240-fold higher in liver and 115-fold higher in spleen than that of R8-MEND alone, demonstrating the

In a similar approach, the pH-sensitive fusogenic peptide GALA was incorporated into a MEND system as an endosomal escape enhancer in a system that comprised of R8 and an MMP-cleavable PEG functionality. Increased gene silencing effect was observed for delivery of anti-luciferase siRNA in HeLa-luc cells in vitro when compared to an unmodified MEND [149]. In addition to this, an in vivo study using a HT1080-luc xenografted model demonstrated that the cleavable PEGylated GALA/R8/MEND exhibited efficient luciferase gene knockdown

Incorporation of targeting motifs has also proved useful in MEND systems. The addition of RGD peptide to MEND, which is a targeting ligand for integrins, resulted in significant tumour growth delay in OS-RC-2 human renal carcinoma bearing mice when RGD-MEND was used to deliver anti-VEGF siRNA in vivo [150]. GALA peptide was also used as a targeting ligand for sialic acid-terminated sugar chains on pulmonary endothelium as reported by Kusumoto et al. [151]. Following intravenous administration of GALA-MEND delivering antiCD31 siRNA in vivo, approximately 50% inhibition of lung metastasis in a Murine melanoma B16- F10 mouse model was observed when compared with control groups. Examples of MENDs

in comparison to PEG-MEND, which was unable to cause any gene knockdown.

and the various functionalities that have been employed are detailed in Table 1.

**component**

INF7 peptide derived from Nterminal domain of the HA2 protein influenza virus envelope

**Nuclear localisation component**

**Other functional**

Protamine - Luciferase

**Activity**

transgene expression levels 240-fold higher in liver and 115-fold higher in spleen

**groups**

**Lipid Envelop Endosomolytic**

**Name Condensing**

R8/INF7- MEND

**material and nucleic acid cargo**

78 Gene Therapy - Principles and Challenges

Protamine (plasmid DNA for luciferase transgene expression)

Egg

phosphatidylcholine (EPC), cholesterol (Chol), 1,2-dioleoylsn-glycero-3 phosphocholine

densing agent resulted in a 70% silencing effect in transfected COS7 fibroblast cells.

importance of optimising functionality of the MEND system [148].


**Table 1.** MEND non-viral delivery systems, their components and applications to gene therapy

The potential of controlled intracellular delivery using the MEND system was also highlighted by Toriyabe et al. who used stearylated-octahistidne (STR-H8) as a pH-responsive component to facilitate the efficient release of siRNA in the cytoplasm [152]. STR-H8 was used to complex anti-luciferase siRNA and delivered using a conventional R8/GALA functionalised MEND. The authors demonstrated that luciferase gene knockdown was significantly higher in HeLa-GL3 cells treated with the STR-H8 MEND than with a MEND containing stearylated octaar‐ ginine (STR-R8) to condense the siRNA. This may be explained by more efficient decondensation and release of siRNA from STR-H8 in the cytoplasm, which was confirmed by a RiboGreen assay showing siRNA release efficiency from STR-H8 was much higher than siRNA release from STR-R8 at pH7.4 (intracellular pH). It is clear, therefore, that PEGylation and unpackaging of DNA are important considerations in the development of MEND systems, and with further optimisation and characterisation, MENDs have great promise as effective non-viral gene delivery agents.

#### **6.2. Bio-inspired systems**

Viral vectors still remain the most efficient gene therapy delivery vehicles with no non-viral delivery system producing comparable gene delivery potencies. Viruses have evolved naturally to infect and transfer their genetic material into host cells [153]. Understanding the various mechanisms by which viruses elicit delivery of genetic material has led to exploitation of viral peptide motifs by gene therapists and molecular engineers [154]. Functional peptide motifs derived from viruses have been engineered and incorporated into a wide range of bioinspired non-viral delivery systems with great success, thereby benefiting from the viruses' expertise, while circumventing immunogenicity and safety concerns associated with viruses [35, 154]. Peptides are an attractive alternative to polymer and lipid-based non-viral vectors as they are less toxic, easily synthesised, and only weakly activate the complement system therefore enhancing safety [155]. The main peptides of interest are generally classified according to their function, i.e., DNA condensing peptides, cell penetrating peptides, endo‐ somolytic peptides, and nuclear location sequences [156].

#### **6.3. DNA-condensing peptides**

**Name Condensing**

**material and nucleic acid cargo**

80 Gene Therapy - Principles and Challenges

non-viral gene delivery agents.

somolytic peptides, and nuclear location sequences [156].

**6.2. Bio-inspired systems**

**Lipid Envelop Endosomolytic**

**Table 1.** MEND non-viral delivery systems, their components and applications to gene therapy

The potential of controlled intracellular delivery using the MEND system was also highlighted by Toriyabe et al. who used stearylated-octahistidne (STR-H8) as a pH-responsive component to facilitate the efficient release of siRNA in the cytoplasm [152]. STR-H8 was used to complex anti-luciferase siRNA and delivered using a conventional R8/GALA functionalised MEND. The authors demonstrated that luciferase gene knockdown was significantly higher in HeLa-GL3 cells treated with the STR-H8 MEND than with a MEND containing stearylated octaar‐ ginine (STR-R8) to condense the siRNA. This may be explained by more efficient decondensation and release of siRNA from STR-H8 in the cytoplasm, which was confirmed by a RiboGreen assay showing siRNA release efficiency from STR-H8 was much higher than siRNA release from STR-R8 at pH7.4 (intracellular pH). It is clear, therefore, that PEGylation and unpackaging of DNA are important considerations in the development of MEND systems, and with further optimisation and characterisation, MENDs have great promise as effective

Viral vectors still remain the most efficient gene therapy delivery vehicles with no non-viral delivery system producing comparable gene delivery potencies. Viruses have evolved naturally to infect and transfer their genetic material into host cells [153]. Understanding the various mechanisms by which viruses elicit delivery of genetic material has led to exploitation of viral peptide motifs by gene therapists and molecular engineers [154]. Functional peptide motifs derived from viruses have been engineered and incorporated into a wide range of bioinspired non-viral delivery systems with great success, thereby benefiting from the viruses' expertise, while circumventing immunogenicity and safety concerns associated with viruses [35, 154]. Peptides are an attractive alternative to polymer and lipid-based non-viral vectors as they are less toxic, easily synthesised, and only weakly activate the complement system therefore enhancing safety [155]. The main peptides of interest are generally classified according to their function, i.e., DNA condensing peptides, cell penetrating peptides, endo‐

**component**

**Nuclear localisation component**

**Other functional**

**Activity**

following intravenous administration of GALA-MEND delivering anti-CD31 siRNA *in vivo* [151].

**groups**

Cationic peptides containing lysine or arginine residues interact electrostatically with the negatively charged phosphate backbone of DNA, condensing and packaging DNA into complexes with a net positive charge, which protects DNA from degradation and allows interaction with cell membranes [157]. Examples include histones, including H2A, which are natural basic proteins [158], µ (mu) peptide derived from adenovirus [159], and TAT peptide from HIV-1 [160]. Condensing peptides alone have a limited role because they cannot over‐ come many of the barriers to gene delivery; although some peptides, such as TAT, have cell penetrating properties, which makes them more attractive options in vector development [161]. However, the unpredictable nature of interactions between peptides and nucleic acids remains an issue and further research is needed for optimisation of vectors [157].

#### **6.4. Cell Penetrating Peptides (CPPs)**

Cell penetrating peptides (CPPs) are generally short peptides that have the ability to cross the cell membrane via various mechanisms including endocytic pathways or through direct translocation, without the need for receptors or other carriers [162]. Such peptides have been shown to deliver various cargoes to a range of cell types; peptide sequences are easily modifiable to optimise properties such as cargo transport or subcellular targeting [163]. Natural peptides exhibiting this penetrating activity include Penetratin (RQI‐ KIYFQNRRMKWKK), derived from the third helix of the homeodomain of Antennapedia [164], and TAT (GRKKRRQRRR) derived from HIV-1 [41]. Both have regions of basic amino acids and an alpha-helical conformation with the ability to translocate a cargo across cell membranes, which highlights the potential application of CPPs in gene therapy. Novel CPPs have since been derived and include peptides with a wide range of structures and characteristics; however, generally CPPs are cationic/basic, amphipathic, or hydrophobic in nature [156].

Amphipathic peptides are composed of both hydrophobic and hydrophilic domains in primary or secondary conformation. The secondary structure produces an alpha-helical structure with the hydrophobic residues such as leucine, glycine, or tryptophan localised on one face of the helix and the hydrophilic residues such as lysine, arginine, or histidine localised on the other. This amphipathic structure has been shown to be essential for passage across the cell membranes [165]. Structural changes of amphipathic peptides contribute to their binding affinity for cell membranes, and insertion of hydrophobic portions of the peptide into the membrane are important for interaction with the lipid membrane and subsequent uptake [54]. However, although amphipathic peptides have shown much promise, not all CPPs rely on this amphipathic nature for internalisation. For example, in the case of Penetratin, it is the positive charges rather than the helical structure that is responsible for cell penetration [166]. Therefore, increasingly, attention has been paid to developing simple linear peptides rich in cationic amino acids such as arginine. Cationic CPPs are composed mainly of basic amino acid residues including arginine, lysine, and histidine [167] and electrostatically bind to various anionic species present on the extracellular surface of the cell membranes, e.g., lipid head groups or proteoglycans such as heparin sulphate [168].

It has been reported that peptides containing arginine residues have stronger cell penetrating ability than peptides comprising lysine and histidine, with the guanidine moiety possessed by arginine being held as crucial for cell entry [169]. As a result, arginine-rich peptides have been extensively researched in order to characterise their activity [170–172]. The discovery that the basic portion of TAT responsible for the cell penetrating activity is rich in arginine residues prompted much research into the characterisation of mechanisms involved in the cellular entry of arginine-rich peptides [41]. It has been elucidated that the exact peptide sequence involved is not as crucial as the length of sequence and number of arginine residues incorporated, with between 6 and 15 arginine residues required for optimum activity [54]. In a study carried out by Wender et al., it was observed that truncated versions of TAT with arginine residues replaced with alanine exhibited reduced cellular uptake, but a 9-mer oligoarginine peptide (R9) was 20-fold more efficient than TAT [173]. Further to this, Mitchell et al. used peptides composed of multiple arginine residues termed oligoarginines, labelled with fluorescein to demonstrate that negligible cell uptake was exhibited with fewer than 6 arginine residues, but that when peptides of 7 arginines or more were tested, fluorescence increased as a function of peptide length up to 15 arginine residues, beyond which no increase in fluorescence was observed. Peptides containing more than 15 arginine residues can still penetrate cells, although this happens at a reduced efficiency and with toxicity to cells [169].

CPPs have the ability to enter any cell they come in contact with and this lack of specificity is problematic for gene therapy [174]. The use of 'smart' delivery vectors with 'activatable' CPPs (ACPs) has been explored, where a CPP is connected to a neutralising polyanion via a cleavable linker, reducing the overall charge and non-specific electrostatic uptake by cells. Enzymes produced in cancerous cells, such as MMPs, can then cleave the linker and allow the CPP to enter cancer cells [175]. For example, Mei et al. reported an ACP that includes a masking sequence of anionic E8 (sequence: EEEEEEEE) to shield the cationic nature of R8 [176]. The mask was linked to R8 by a MMP-2 sensitive linker; when the ACP nanoparticles were in the tumour environment, which overexpresses MMP-2, the mask was cleaved exposing R8 to tumour cells allowing tumour specific uptake. The authors used in vivo imaging to demon‐ strate this, while also showing lower ACP nanoparticle distribution in other tissues.

Another strategy for targeting and cell specificity has focused on the use of cell-penetratinghoming peptides (CPHPs) [97, 177] that combine targeting and cell penetration abilities. Kondo et al. described a CPHP known as RLW (peptide sequence: RLWMRWYSPRTRAYG) found through systematic selection from a random peptide library that had the ability to selectively target and penetrate A549 non-small cell lung cancer cells via an unknown mechanism thought to involve specific RLW ligand receptors on A549 cells [178]. Gao et al. demonstrated that when RLW was anchored onto poly(ethyleneglycol)-poly(ε-caprolactone) (PEG-PCL) nanoparticles loaded with infrared dye (DiR) cellular uptake was 2-fold higher in A549 cells than in umbilical vein endothelial cells in vitro [179]. Further to this, in vivo imaging showed the RLW nano‐ particles targeted A549 xenografts specifically over U87 xenografts, with only low levels seen in normal organs in comparison to PEG-PCL nanoparticles functionalised with R8, which evoked DiR accumulation in all tissues. The specificity of CHCPs is a great asset; however, elucidation of the exact mechanism of how CHCPs work and a broader spectrum of activity may be more attractive so that a peptide may be used to treat more than one cancer type.

The cargo being carried by the vector must also be considered when designing a vector, as CPPs interact with various cargoes in different ways. For example, TAT mediates internalisa‐ tion by at least two distinct pathways. Large cargoes, e.g., proteins, enter via caveolae endo‐ cytosis and macropinocytosis leading to endosomal entrapment, whereas small cargoes, e.g., peptides, enter slowly by endocytosis and rapidly by transduction by an unknown mechanism that gives direct access to the cytosol [42]. As endosomal entrapment is a major barrier to transfection, CPPs have been functionalised with endosomolytic peptides. Liou et al. described a fusion peptide that combines R9 for cell penetration and hemagglutinin-2 (HA2) for endo‐ somal escape; the resulting vector was tagged with red fluorescent protein (RFP) for imaging purposes [180]. Significantly more RFP was detected in vitro when A549 human lung carci‐ noma cells were treated with the R9-HA2 peptide in comparison to R9 alone.

Problems with CPPs, such as humoral immune response induction, as seen in studies with Penetratin [181], and stability need to be addressed. Amino acids exist in different isoforms with variable susceptibility to degradation by proteases in serum. The L-isoform found in abundance in nature is sensitive to degradation, but the D-isoform is more resistant due to the altered stereochemistry that affects protease recognition. The use of the D-isoform of amino acids has therefore been suggested as a modification to render CPPs protease-resistant, enhancing stability [182]. However, further characterisation of the structure-activity relation‐ ship of individual CPPs is needed to allow the tailoring of specific CPPs to particular intra‐ cellular targets, optimising efficiency and reducing side effects [183].

#### **6.5. Endosomolytic peptides**

It has been reported that peptides containing arginine residues have stronger cell penetrating ability than peptides comprising lysine and histidine, with the guanidine moiety possessed by arginine being held as crucial for cell entry [169]. As a result, arginine-rich peptides have been extensively researched in order to characterise their activity [170–172]. The discovery that the basic portion of TAT responsible for the cell penetrating activity is rich in arginine residues prompted much research into the characterisation of mechanisms involved in the cellular entry of arginine-rich peptides [41]. It has been elucidated that the exact peptide sequence involved is not as crucial as the length of sequence and number of arginine residues incorporated, with between 6 and 15 arginine residues required for optimum activity [54]. In a study carried out by Wender et al., it was observed that truncated versions of TAT with arginine residues replaced with alanine exhibited reduced cellular uptake, but a 9-mer oligoarginine peptide (R9) was 20-fold more efficient than TAT [173]. Further to this, Mitchell et al. used peptides composed of multiple arginine residues termed oligoarginines, labelled with fluorescein to demonstrate that negligible cell uptake was exhibited with fewer than 6 arginine residues, but that when peptides of 7 arginines or more were tested, fluorescence increased as a function of peptide length up to 15 arginine residues, beyond which no increase in fluorescence was observed. Peptides containing more than 15 arginine residues can still penetrate cells, although

CPPs have the ability to enter any cell they come in contact with and this lack of specificity is problematic for gene therapy [174]. The use of 'smart' delivery vectors with 'activatable' CPPs (ACPs) has been explored, where a CPP is connected to a neutralising polyanion via a cleavable linker, reducing the overall charge and non-specific electrostatic uptake by cells. Enzymes produced in cancerous cells, such as MMPs, can then cleave the linker and allow the CPP to enter cancer cells [175]. For example, Mei et al. reported an ACP that includes a masking sequence of anionic E8 (sequence: EEEEEEEE) to shield the cationic nature of R8 [176]. The mask was linked to R8 by a MMP-2 sensitive linker; when the ACP nanoparticles were in the tumour environment, which overexpresses MMP-2, the mask was cleaved exposing R8 to tumour cells allowing tumour specific uptake. The authors used in vivo imaging to demon‐

strate this, while also showing lower ACP nanoparticle distribution in other tissues.

Another strategy for targeting and cell specificity has focused on the use of cell-penetratinghoming peptides (CPHPs) [97, 177] that combine targeting and cell penetration abilities. Kondo et al. described a CPHP known as RLW (peptide sequence: RLWMRWYSPRTRAYG) found through systematic selection from a random peptide library that had the ability to selectively target and penetrate A549 non-small cell lung cancer cells via an unknown mechanism thought to involve specific RLW ligand receptors on A549 cells [178]. Gao et al. demonstrated that when RLW was anchored onto poly(ethyleneglycol)-poly(ε-caprolactone) (PEG-PCL) nanoparticles loaded with infrared dye (DiR) cellular uptake was 2-fold higher in A549 cells than in umbilical vein endothelial cells in vitro [179]. Further to this, in vivo imaging showed the RLW nano‐ particles targeted A549 xenografts specifically over U87 xenografts, with only low levels seen in normal organs in comparison to PEG-PCL nanoparticles functionalised with R8, which evoked DiR accumulation in all tissues. The specificity of CHCPs is a great asset; however, elucidation of the exact mechanism of how CHCPs work and a broader spectrum of activity may be more attractive so that a peptide may be used to treat more than one cancer type.

this happens at a reduced efficiency and with toxicity to cells [169].

82 Gene Therapy - Principles and Challenges

The harsh endosomal environment can lead to degradation of peptides and their cargo, as CPPs, such as TAT and oligoarginines, lack the ability to escape the endosome unaided, resulting in poor transfection efficiencies [184]. Histidine-rich peptides are usually endoso‐ molytic in nature and can facilitate endosomal escape through the proton sponge where the protonation of imidazole groups in histidine-rich peptides facilitates buffering of the endo‐ some causing endosomes to swell and burst, releasing their contents [185, 186]. Another mechanism employed by histidine-rich peptides is the 'flip-flop' effect, which may operate depending on the number of histidine residues or their arrangement in a peptide [187]. In a study conducted by Lo et al., the addition of 10 histidine residues to TAT increased luciferase transgene expression up to 7,000-fold in the human glioma cell line U251 in vitro [188]. Bafilomycin A1, a known inhibitor of the proton sponge effect for endosomal escape, in turn inhibited transfection significantly, supporting the idea that the activity of histidine as an endosomal escape motif could improve the transfection efficiency of TAT. However, in vivo administration of the TAT-histidine peptide/DNA complexes to deliver the luciferase reporter gene into the brain of rats showed 5-fold lower expression than was achieved using PEI 25 kDa/DNA complexes, suggesting more work needs to be done to ensure in vitro results translate to the in vivo setting. One example of a histidine-rich peptide that has shown great promise is H5WYG, derived from the HA2 subunit of haemaglutinin (HA) protein of the influenza virus. H5WYG causes endosomal escape through the proton sponge effect, when the histidine residues become protonated at around pH 6. H5WYG is unaffected by the presence of serum that gives it an added advantage of being suitable for in vivo gene delivery [189]. Asseline et al. reported a 2-fold increase in luciferase mRNA levels when H5WYG was added to an antisense oligonucleotide (2'-Ome RNA705) targeting aberrant splicing of luciferase pre-mRNA in HeLa pLuc705 cells [190].

Fusogenic peptides have also been of great importance in facilitating endosomal escape [191]. Pore formation may be mediated by cationic amphiphilic peptides that bind to the lipid bilayer of the endosomal membrane, causing internal stress or tension leading to pore formation. Fusogenic peptides are known to adopt an amphipathic α-helical structure when pH drops to around 5 within the endosome, causing interaction with the phospholipid membrane and endosomal disruption [156, 192]. This fusogenic activity also allows these peptides to interact with cell membranes and facilitate internalisation, giving some fusogenic peptides a dual function with ability to package nucleic acid to avoid degradation and be delivered into the cytoplasm of the cell. One such example is RALA (WEARLARALARALARHLARALARAL‐ RACEA), a 30 amino acid fusogenic peptide with a cationic nature [193]. It is composed of a hydrophilic arginine (R) region that facilitates condensation of anionic complexes, e.g., DNA; a hydrophobic leucine (L) region that interacts with lipid membranes; and an alanine (A)-rich region that gives the peptide amphipathicity. This structure allows RALA to maintain α-helical conformation at low pH, enabling endosomal escape. The design of RALA was informed by the understanding of two similar peptides, namely GALA (WEAALAEALAEALAEHLAEA‐ LAEALEALAA) and KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), peptides that were in turn derived from the HA2 subunit of the influenza virus, with GALA being the first cell penetrating amphipathic peptide demonstrated to possess fusogenic activity [192]. However, GALA carries an overall negative charge and therefore cannot be used for delivery of DNA alone. KALA was derived by substituting the glutamic acid (E) in GALA with lysine (K); the resulting derivative was positively charged, and thereby more suitable for delivery of DNA. This E to K substitution resulted in improved interaction with negatively charged cell membranes and allowed condensation of negatively charged DNA cargoes [194]. RALA was derived by substituting lysine residues with arginine (R), which conferred a lower toxicity [172, 193].

#### **6.6. Nuclear Localisation Sequences (NLSs)**

Intracellular trafficking of nucleic acid cargo and entry into the nucleus is crucial for transgene expression. The use of nuclear localisation sequences (NLS) has proved beneficial in improving the efficiency of vectors. NLSs help traffic vectors towards the nucleus and facilitate entry through the nuclear envelope in association with the importin pathway [62]. Classical nuclear localisation signals, such as the NLSs from simian virus 40 (SV40), large tumour antigen (PKKKRKV), and Rev peptide (RRNRRRRWRERQRQ), consist of short stretches of basic amino acids [195]. Such NLSs have the ability to bind DNA in order to facilitate nuclear entry. Elder et al. used atomistic molecular dynamics to investigate the effect of peptide chemistry and sequence on DNA binding behaviour, focusing on the NLS from SV40 [196]. By analysing the conformational entropy and free energy of binding, the authors found that replacing arginine with lysine reduced binding strength by eliminating arginine–DNA interactions, but placing arginine in a less sterically hindered location has little effect on polycation–DNA binding strength. This strong binding ability of arginine is important for an NLS because nucleic acids need to be bound and protected from degradation by nucleases in the cytosol before reaching the nucleus.

presence of serum that gives it an added advantage of being suitable for in vivo gene delivery [189]. Asseline et al. reported a 2-fold increase in luciferase mRNA levels when H5WYG was added to an antisense oligonucleotide (2'-Ome RNA705) targeting aberrant splicing of

Fusogenic peptides have also been of great importance in facilitating endosomal escape [191]. Pore formation may be mediated by cationic amphiphilic peptides that bind to the lipid bilayer of the endosomal membrane, causing internal stress or tension leading to pore formation. Fusogenic peptides are known to adopt an amphipathic α-helical structure when pH drops to around 5 within the endosome, causing interaction with the phospholipid membrane and endosomal disruption [156, 192]. This fusogenic activity also allows these peptides to interact with cell membranes and facilitate internalisation, giving some fusogenic peptides a dual function with ability to package nucleic acid to avoid degradation and be delivered into the cytoplasm of the cell. One such example is RALA (WEARLARALARALARHLARALARAL‐ RACEA), a 30 amino acid fusogenic peptide with a cationic nature [193]. It is composed of a hydrophilic arginine (R) region that facilitates condensation of anionic complexes, e.g., DNA; a hydrophobic leucine (L) region that interacts with lipid membranes; and an alanine (A)-rich region that gives the peptide amphipathicity. This structure allows RALA to maintain α-helical conformation at low pH, enabling endosomal escape. The design of RALA was informed by the understanding of two similar peptides, namely GALA (WEAALAEALAEALAEHLAEA‐ LAEALEALAA) and KALA (WEAKLAKALAKALAKHLAKALAKALKACEA), peptides that were in turn derived from the HA2 subunit of the influenza virus, with GALA being the first cell penetrating amphipathic peptide demonstrated to possess fusogenic activity [192]. However, GALA carries an overall negative charge and therefore cannot be used for delivery of DNA alone. KALA was derived by substituting the glutamic acid (E) in GALA with lysine (K); the resulting derivative was positively charged, and thereby more suitable for delivery of DNA. This E to K substitution resulted in improved interaction with negatively charged cell membranes and allowed condensation of negatively charged DNA cargoes [194]. RALA was derived by substituting lysine residues with arginine (R), which conferred a lower toxicity

Intracellular trafficking of nucleic acid cargo and entry into the nucleus is crucial for transgene expression. The use of nuclear localisation sequences (NLS) has proved beneficial in improving the efficiency of vectors. NLSs help traffic vectors towards the nucleus and facilitate entry through the nuclear envelope in association with the importin pathway [62]. Classical nuclear localisation signals, such as the NLSs from simian virus 40 (SV40), large tumour antigen (PKKKRKV), and Rev peptide (RRNRRRRWRERQRQ), consist of short stretches of basic amino acids [195]. Such NLSs have the ability to bind DNA in order to facilitate nuclear entry. Elder et al. used atomistic molecular dynamics to investigate the effect of peptide chemistry and sequence on DNA binding behaviour, focusing on the NLS from SV40 [196]. By analysing the conformational entropy and free energy of binding, the authors found that replacing arginine with lysine reduced binding strength by eliminating arginine–DNA interactions, but

luciferase pre-mRNA in HeLa pLuc705 cells [190].

84 Gene Therapy - Principles and Challenges

[172, 193].

**6.6. Nuclear Localisation Sequences (NLSs)**

Several other proteins derived from viruses are excellent at traversing the intracellular network and facilitating nuclear import [197]. The TAT, Rev, and Rex proteins of the retroviruses contain arginine-rich NLSs, which have the ability to shuttle to and from the nucleus. Herpes simplex virus (HSV) type 1 tegument proteins, known as VP13/14, are also arginine-rich and act in a similar way [198, 199]. The arginine-rich portion of these proteins is responsible for the nuclear import, with leucine-rich portions, known as nuclear export signals (NES), being responsible for the shuttling between the nucleus and cytoplasm. Arginine-rich NLSs have been shown to use importin β pathway with no involvement of importin α pathway [200]. Importin β is not only involved in nuclear import but is also a potential adaptor for movement along microtubules, which may enhance trafficking of arginine-rich peptides to the nucleus, as well as entry to the nucleus [201]. Identification of exact binding sites and utilisation of such mechanisms may be the key to improving transfection efficiencies for peptide delivery vectors.

Incorporation of such sequences has proven to be useful in vector design. Hatefi et al. dem‐ onstrated that the addition of Rev (RRNRRRRWRERQRQ) to their fusion peptide KALA-2H1- NLS-TP facilitated cargo delivery to the nucleus by utilising microtubules for nuclear localisation [186]. Non-classical NLSs, such as M9 from human mRNA binding protein hnRNP A1, have also shown promise for non-viral vector functionalisation [202]. These NLSs lack stretches of basic amino acids and do not enter the nucleus via the importin pathway. M9 binds to the transportin receptor that results in nuclear localisation and has shown the ability to transport the vector towards the nucleus by shuttling between the nucleus and cytoplasm [203–205]. These properties make M9 an attractive NLS for gene delivery [65]. A number of viruses are known to exploit host microtubule machinery to facilitate access to the nucleus [206], but little is known about the exact mechanisms and binding domains used by viruses, and further study is required to elucidate exact peptide sequences involved that may be incorporated into non-viral vectors for rational design to achieve enhanced transfection efficiencies [207]. For example, the motif sequence contained in the adenoviral capsid hexon (E3-14.7K peptide: VVMVGEKPITITQHSVETEG) was conjugated to plasmid DNA and promoted microtubule-mediated transport of the DNA, resulting in 2.5-fold increase in transfection efficiency in HeLa cells compared to plasmid DNA only [208]. Incorporation of this sequence into a non-viral vector may therefore improve transfection efficiency.

Problems have been encountered where binding of an NLS with DNA renders the NLS unable to bind to the importins that allow passage through the NPC. Using a basic NLS to condense and deliver DNA alone has not been successful because they do not bind DNA strongly enough and the complexes are generally broken down in the cytoplasm [209]. Covalent conjugation of an NLS to DNA has been problematic as this may render the NLS or the DNA non-functional, as demonstrated when covalent bonding of SV40 did not increase nuclear localisation of pDNA [68]. Various binding strategies have been used to improve this, as well as using condensing agents such as histones that also possess nuclear localisation properties [210], but generally, NLSs are used to supplement other delivery systems rather than as stand-alone vectors.

#### **6.7. Designer Biomimetic Vectors (DBVs)**

An exciting approach to the multifunctional vector has been the introduction of recombinant production of bio-inspired fused protein sequences, each coding for a discrete motif with an explicit barrier evasion function [211]. Termed designer biomimetic vectors, these vectors are rationally designed to incorporate several motifs with distinct functions, and could be a step towards the production of 'artificial viruses'. The previous strategies discussed involving different components of a multi-functional system being conjugated together by various attachments may not be ideal for production of gene delivery systems. Simple conjugation of certain peptides has also led to alteration in the function of the peptides [212], therefore conjugating all the desired components together may be problematic. Production of DBVs using recombinant DNA technology allows the fusion of discrete motifs in a relatively simple process that should not affect the functional operation of the motifs. This would circumvent any problems involved with complex conjugation reactions to attach different components and ultimately could be more cost effective and reproducible in a large scale industrial setting. The production process involves introduction of plasmids, which have been engineered to contain the desired motifs for the protein, into competent bacterial cells. The bacteria then utilise the plasmid to produce the fusion proteins, which are subsequently extracted and purified. The use of this recombinant DNA technology allows the specific design of the vector at the molecular level, which can be tailored to enhance and optimise gene delivery [213]. Examples of multi-functional recombinant vectors are detailed in Table 2.



**6.7. Designer Biomimetic Vectors (DBVs)**

86 Gene Therapy - Principles and Challenges

**Name Nucleic acid**

Tetra-H2A(TH)

KALA-2H1- NLS-TP

**condensation**

Four tandem repeats of human histone H2A peptide (TH)

Two repeating units of histone H1 (2H1)

An exciting approach to the multifunctional vector has been the introduction of recombinant production of bio-inspired fused protein sequences, each coding for a discrete motif with an explicit barrier evasion function [211]. Termed designer biomimetic vectors, these vectors are rationally designed to incorporate several motifs with distinct functions, and could be a step towards the production of 'artificial viruses'. The previous strategies discussed involving different components of a multi-functional system being conjugated together by various attachments may not be ideal for production of gene delivery systems. Simple conjugation of certain peptides has also led to alteration in the function of the peptides [212], therefore conjugating all the desired components together may be problematic. Production of DBVs using recombinant DNA technology allows the fusion of discrete motifs in a relatively simple process that should not affect the functional operation of the motifs. This would circumvent any problems involved with complex conjugation reactions to attach different components and ultimately could be more cost effective and reproducible in a large scale industrial setting. The production process involves introduction of plasmids, which have been engineered to contain the desired motifs for the protein, into competent bacterial cells. The bacteria then utilise the plasmid to produce the fusion proteins, which are subsequently extracted and purified. The use of this recombinant DNA technology allows the specific design of the vector at the molecular level, which can be tailored to enhance and optimise gene delivery [213].

Examples of multi-functional recombinant vectors are detailed in Table 2.

GALA peptide N/A (anti-

KALA peptide NLS from Rev

protein of HIV

virus

**Nuclear localisation**

luciferase siRNA delivery)

**Targeting motif**

Anisamide (AA) to target cancer cells that overexpress sigma receptor

ZR-75-1 targeting peptide (RVCFLWQ DGRCVF)

**Other Activity**

TH produced a higher silencing efficiency in HT60-luc cells *in vitro* and *in vivo* than the NPs assembled with protamine as the nucleic acid

condensing agent [218].


PEGylated, cathepsin D cleavage sites in the TH for digestion in endosome compartment, DOTAP and Chol Lipid envelope surrounding

TH

**Endosomolytic component**

**Table 2.** Recombinant multifunctional non-viral delivery systems, their components, and application in gene therapy

Recently the Gandehari and Hatefi groups have reported the design and development of recombinant fusion proteins for targeted gene delivery [34, 211]. The DBVs are produced by fusing the desired motif sequences, usually composed of a DNA condensing motif (DCM), endosomal disruption motif (EDM) and nuclear localisation motif (NLS) [34, 214]. Sadeghian et al. described a fusion protein comprised of two repeats of histone H1 for DNA condensation, H5WYG pH responsive fusogenic peptide for endosomal escape and the simian virus 40 (SV40) large T-antigen NLS for a nuclear localization [211]. The fusion peptide was complexed with the pGL3 plasmid for luciferase expression to form nanoparticles; the nanoparticles transfected Chinese hamster ovary (CHO) cells efficiently in vitro. However, this system lacked a targeting motif that is highly desirable in the design of gene delivery system.

Soltani et al. recently described a delivery system known as KALA-2H1-NLS-TP, which is composed of two repeating units of histone H1 (2H1) to efficiently condense DNA into nanosized particles, a synthetic pH-dependent endosome disrupting motif (KALA) to promote escape from endosomes, a cyclic targeting peptide (TP) selected from a phage display library to target antigens on the surface of ZR-75-1 breast cancer cells, and an NLS from the Rev protein of HIV to facilitate translocation of DNA towards the cell nucleus [186]. The authors demon‐ strated that the recombinant vector had a high rate of gene transfection efficiency compared to vectors that lacked one or more functional motifs, and targeted the ZR-75-1 cells. Besides the ability to target, the developed multifunctional vector was able to disrupt endosomal membranes, reach the nucleus by utilizing microtubules, and transfect efficiently while showing no detectable toxicity. McCarthy et al. presented similar results using a DBV for the delivery of iNOS gene therapy targeted to breast cancer [215].

Canine et al. described a biopolymer termed FP–(DCE)*n*–NLS–CS–TM that contains repeating units of arginine and histidine to condense pDNA and lyse endosome membranes (DCE), a HER-2 targeting affibody to target cancer cells (TM), a pH responsive fusogenic peptide (FP) H5WYG to destabilize endosome membranes and enhance endosomolytic activity of histidine residues, and a nuclear localization signal (NLS) M9 to enhance translocation of pDNA towards the cell nucleus. A cathepsin D enzyme substrate (CS) was also engineered in between targeting motif and NLS to facilitate dissociation of the targeting motif from the biopolymer inside late endosomes where cathepsin D is abundant [216]. The authors demonstrated the functioning of each motif in the polymer resulting in successful transfection of SKOV-3 and GFP transgene expression.

The production of these recombinant vectors renders it relatively easy to change their charac‐ teristics by sequence modification. Canine et al. further demonstrated that by modifying the sequence of the biopolymer FP–(DCE)*n*–NLS–CS–TM, it was possible to fine tune the vector for either delivery of plasmid DNA to the nucleus or delivery of siRNA to the cytoplasm [217]. It was reported that inclusion of the M9 NLS rendered the biopolymer (FP–(DCE)*n*–NLS–CS– TM) suitable for delivery of plasmid DNA to the nucleus but not for delivery of siRNA. However, exclusion of the NLS from the biopolymer (FP– (DCE)*n*–CS–TM) rendered it more suitable for delivery of siRNA to the cytoplasm but not for nuclear delivery of plasmid DNA in SKOV-3 human ovarian cancer cells. This study demonstrates the possibility of not only targeting specific cells for gene delivery, but also the ability to target intracellular compart‐ ments depending on the nature of the therapeutic to be delivered.

Wang et al. presented a novel recombinant protein tetra-H2A (TH) derived from histone H2A that was developed to replace protamine as a conditionally reversible, nucleic acid condensing agent. The recombinant protein comprised of four tandem repeats of human histone H2A peptide, interspersed with cathepsin D cleavage sites and a pH-responsive fusogenic peptide GALA to facilitate the endosome escape of the cargo. The recombinant protein, tetra-H2A (TH), was able to condense siRNA into a stable complex that was in turn coated in a cationic lipid with a high degree of PEGylation, forming Lipid-tetra-H2A-Hyaluronic acid (LHH) nanopar‐ ticles [218]. This design was developed in order to mimic lipid-enveloped viruses to replicate the transfection abilities of viruses in vivo. The histone-containing polymer demonstrated an enhanced intracellular release of the cargo and an increased anti-luciferase siRNA silencing efficiency in vitro compared with the protamine-containing polymer in H460-luc human lung carcinoma cells. Furthermore, in vivo gene silencing by tumour-targeted anti-luciferase siRNA was evaluated in H460-luc xenograft-bearing mice with the histone-containing nanoparticles loaded with anti-luciferase siRNA resulting in ~66% silencing of luciferase expression, significantly higher than that of the protamine-mediated knockdown (34%). This study demonstrates the importance of efficient release of the genetic payload for efficient gene therapy; through optimisation of each component of the multifunctional vector, it may be possible to maximise transfection efficiency. However, these vectors are still in the early stage of development and much research is needed. Many of these findings serve only to confirm the theory behind the design of the vector, and further in vivo work with therapeutic trans‐ genes is ultimately required.
