**3.1 Physical systems**

Gene delivery systems can be classified based on their means of achieving transfection (Figure 2). There are systems that utilise physical forces, mostly with the aim of disrupting the plasma membrane, to facilitate nucleic acids delivery. For instance, electroporation and sonoporation (Frenkel, 2008) uses electrical and sonication forces respectively to transiently compromise the plasma membrane. The ballistic gene gun method, on the other hand,

Fig. 2. The different classes and examples of gene delivery systems. This chapter will concentrate on peptide-derived vectors.

directly shoots DNA-coated metal particles (frequently gold) into cells (Merediz et al., 2000). Expectedly, such aggressive methods can irreversibly damage the cell membrane and cause widespread cell death. Scrape loading, first described in 1984, requires the forceful scraping of cells attached to their culture dishes, thereby creating pores on their membranes for plasmid entry (McNeil et al., 1984). This technique, however, is only applicable to adherent cells. The osmotic lysis of pinosomes was proposed in 1982 and requires that cells capable of pinocytosis be initially exposed to a hypertonic medium of sucrose, PEG and the plasmid of interest (Okada & Rechsteiner, 1982). Subsequent exchange to a hypotonic medium then released the pinosomal content. Although cells are constitutively capable of pinocytosis, such wild fluctuations in osmotic conditions can cause cell death. Finally, microinjection refers to the piecemeal injection of plasmids directly into the cell. This technique, while relatively gentle, is extremely laborious. As such, it is prone to failure and the number of cells that can be processed is limited.

### **3.2 Viral vectors**

620 Non-Viral Gene Therapy

expression was simply accepted to be due to the inclusion of NLS. Given that most NLS are cationic, it is debatable if the observed increase in transfection is strctly the result of improved nuclear import *per se*, or due to other non-specific effects such as enhanced plasmid association and uptake. Moreover, the fact that a NLS can be sterically hindered by plasmids upon binding also contradicts the requirement of NLS to be freely accessible for

In light of the many obstacles that nucleic acids and their carriers have to surmount, it is perhaps understandable that less than 10% of the pool of plasmids that made it into a cell will go on to accumulate in the nucleus (Lam & Dean, 2010). The challenge is to design a multifunctional vector that can address the issues highlighted above and yet, remain safe to

Gene delivery systems can be classified based on their means of achieving transfection (Figure 2). There are systems that utilise physical forces, mostly with the aim of disrupting the plasma membrane, to facilitate nucleic acids delivery. For instance, electroporation and sonoporation (Frenkel, 2008) uses electrical and sonication forces respectively to transiently compromise the plasma membrane. The ballistic gene gun method, on the other hand,

Fig. 2. The different classes and examples of gene delivery systems. This chapter will

concentrate on peptide-derived vectors.

interaction with importins, the nuclear entry regulating proteins.

**3. Overview and classification of gene delivery systems** 

use in a human body.

**3.1 Physical systems** 

There are systems that function as particulate carriers by ferrying nucleic acids into or near to their site of action. Such systems can be viral or non-viral in nature. Viral vectors such as adenovirus, adeno-asociated virus (AAV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), herpes simplex virus (HSV) and murine leukaemia virus (MLV) have been used and comprehensively reviewed elsewhere (Kay et al., 2001). The prime advantage of viral vectors is their transfection efficiency which has benefitted from centuries of selective evolutionary pressure. As a result, viruses are several orders of magnitude more efficient than non-viral vectors (Kircheis et al., 1997) and account for about 70% of all clinical trials involving gene therapy so far (Won et al., 2009). A recent success for viral gene therapy involved the use of lentiviruses to deliver a correct copy of a therapeutic gene to an adult patient suffering from β-thalassaemia (Cavazzana-Calvo et al., 2010). Such patients suffer from defective haemoglobin production and require chronic blood transfusion for survival. Upon reinfusing the patient with his own bone-marrow haematopoietic stem cells that had earlier been transduced *ex vivo*, the patient became transfusion free 1 year after treatment and has been doing well, according to the most recent report at 33 months after treatment. The longer-term outcome, of course, remains to be seen. Having said that, investigators have not abandoned all forms of non-viral research as there are limitations in the use of viral vectors. For example, the technical difficulty of scaling up virus production compliant to good manufacturing practices (GMP) may prevent such therapy from being cheaply accessible (Sheridan, 2011). The size of the construct that can be delivered is also limited. Above all, toxicity and immunogenicity (reviewed by Nayak & Herzog, 2010) provoked by the viral vectors can and have brought clinical trials to premature ends. Repeated administration is also not possible if the body has mounted a systemic immune response. The danger of viral gene therapy was first illustrated about a decade ago by the death of Jesse Gelsinger (Hollon, 2000). Researchers were using experimental adenoviruses to correct his partial ornithine transcarbamylase deficiency affecting the ability of his body to metabolise nitrogen. Unfortunately, the systemic inflammatory response syndrome was triggered and he succumbed, rather rapidly, to multiple organ failure. In another high-profile example, stem cells transduced *ex vivo* with MLV were used to treat children suffering from X-linked severe combined immunodeficiency (Hacein-Bey-Abina et al., 2003). However, MLV is an integrating retrovirus and can cause insertional mutagenesis, which is a phenomenon where random

Peptides as Promising Non-Viral Vectors for Gene Therapy 623

BSA were also delivered into human fibroblast cells using PEG as a fusogen (Wojcieszyn et al., 1981). Dehydration may play a role in PEG-mediated fusion and intriguingly, purification by recrystallisation removed the fusogenic ability of PEG, suggesting that other

Plasmids were initially delivered by synthetic liposomes using concepts adapted from RBC technology. In an early study, plasmids were encapsulated within anionic liposomes and fused with tobacco mesophyll protoplasts using PEG (Deshayes et al., 1985). Since 1987, however, surface binding became popular when liposomes made of *N*-[1-(2,3, dioleyloxy)propyl]-*N*,*N*,*N*-trimethylammonium chloride (DOTMA) were synthesised for the first time (Felgner et al., 1987). DOTMA contains cationic quaternary ammonium groups which can be used for DNA binding. Lipofectin®, the prototypical lipid formulation and a workhorse for transfection experiments today, is a 1:1 mixture of DOTMA and the neutral lipid, dioleoyl phosphatidylethanolamine (DOPE). In this formulation, DOPE functions as a fusogen to aid cellular uptake. Several other formulations of liposomes have been reported. Gao and Huang prepared liposomes made out of DOPE and a novel cationic cholesterol derivative, 3*β*-[*N*-(*N'*,*N'*-dimethylamino)ethane-carbamoyl] cholesterol (DC-chol), and used it to transfect several cell lines with high efficiency (Gao & Huang, 1996). A mixture of *N*′,*N*′ dioctadecyl-*N*-4,8-diaza-10-aminodecanoylglycine amide (DODAG) and DOPE was also proposed to be a novel liposomal formulation that is good for both DNA and siRNA delivery (Mével et al., 2010). In another study, the cationic lipid 1,2-dilinoleyloxy-3 dimethylaminopropane (DLinDMA) was used as a starting template and systematically modified to yield an optimised construct (Semple et al., 2010). Although toxicity remains one of the chief complaints, liposomes are definitely one of the most successful non-viral vectors around. Already, several sustained-release liposomal drug formulations have been approved for human use and thermosensitive liposomes are being evaluated in clinical trials (Hossann et al., 2010). Lipids also dominate in gene therapy trials aimed at using non-viral

An advantage of polymeric carriers is that they offer so much structural diversity that their potential should, theoretically, be limited only by the imagination of chemists. Two of the more archaic examples including PVP and PVA have been mentioned. Poly(D,L-lactic-coglycolic acid) (PLGA), a polymer originally developed for controlled drug release, has also been used to encapsulate plasmids for delivery (Wang et al., 1999). Most polymeric carriers, however, are cationic in nature. Dendritic poly(amido amine) (PAMAM) which contained primary amines for DNA binding was further decorated with PEG and sugars (Wood et al., 2005) or peptide (Wood et al., 2008) for targeting purposes. Linear PAMAM was functionalised with disulfide bonds to make the polymer biodegradable and various chemical side-groups were attached to test for its impact on transfection efficiency (Lin et al., 2007). A large library of poly(β-amino ester)s has been synthesised and systematically screened for transfection efficiency (Green et al., 2006). The effects of molecular weight and charge density of polyphosphoramidates (PPA) on DNA binding and buffering capacity have been reported (Ren et al., 2010). A series of novel enzyme-degradable polycarbonates (PC) with various aliphatic side-chains attached were prepared and shown to effectively transfect cells with virtually no toxicity (Seow & Yang, 2009a). Poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA) was also shown to transfect cells with high efficiency (Lin et al., 2008). Indeed, there are so many other classes of polymers being developed that it is impossible to do all justice with this paragraph. Nonetheless, amongst all, the most notable polymer must be PEI. PEI was first developed in 1995 (Boussif et al.,

ingredients in commercial PEG was essential (Wojcieszyn et al., 1983).

vectors to treat cystic fibrosis (Griesenbach & Alton, 2009).

viral integration can trigger the activation of an oncogene or disrupt genes with tumorsuppressive properties. Consequently, several patients became leukemic and at least one has died so far (Sheridan, 2011). More recently, retroviruses were also used to treat two patients diagnosed with X-linked chronic granulomatous disease which affected the ability of their phagocytes to clear bacterial infections (Ott et al., 2006). Insertional mutagenesis was again responsible for a clone of cells whose genes responsible for growth were activated, prompting fears that such cells may turn cancerous. Having said that, viral gene therapy is not expected to be perfect and its success should be assessed relative to existing treatments. Nonetheless, researchers have not completely mastered the use of viruses and there is still a need for alternative non-viral vectors with safer profiles.

#### **3.3 Non-viral vectors**

Diethylaminoethyl (DEAE)-dextran and calcium phosphate (CaPO4) were two of the earliest systems popular in the 1970s and 80s. DEAE-dextran has a cationic polysaccharide backbone and one of the earliest reports in 1965 used this polymer in a 1:1 mixture with nucleic acids to transfect rhesus monkey kidney cells (Vaheri & Pagano, 1965). Authors of this study also insightfully commented that DEAE-dextran, like histones, could bind and protect nucleic acids from degradation. Although these are now fundamental concepts in modern carrier design, it may not have been as obvious in the past. The co-precipitation of CaPO4 with DNA was first described in 1973, when it was observed that Ca2+ (not Mg2+ or Na+) and PO42- ions, at high enough concentrations, could enhance DNA uptake by cells (Graham & Eb, 1973). This technique was further shown to be sensitive to pH, amount of DNA used and even the level of CO2 in the incubator (Chen & Okayama, 1987). Using an optimised protocol, CaPO4-mediated transfection achieved up to 50% efficiency with a murine L cell line (Chen & Okayama, 1987).

Chitosan is a polysaccharide obtained by the deacetylation of chitin, an exoskeletal component of crustaceans. Each deacetylation site contains a primary amine (pKa~6.5) which allows chitosan to bind nucleic acids. The degree of deacetylation also determines its biodegradability and transfection efficiency. Chitosan was first described as a plasmid carrier by Mumper and colleagues in 1995 and is known to be biocompatible, mucoadhesive and virtually non-toxic (MacLaughlin et al., 1998). As such, chitosan has been evaluated in rabbits, although reporter gene expression in that study was low (MacLaughlin et al., 1998). Strong interactions between high molecular chitosan and DNA were blamed for the overlystable complexes that could not release their plasmid cargos. Using lower molecular weight chitosan, *in vitro* transfection efficiency was improved by up to 24 fold (Koping-Hoggard et al., 2004). Adding histidine (pKa~6) residues to chitosan further improved buffering capacity and overall transfection efficiency (Chang et al., 2010). Recently, it was also shown that the introduction of thiol groups in *N,N,N*-trimethylated chitosan improved its efficiency as a siRNA carrier (Varkouhi et al., 2010).

Red blood cells (RBC) were used for the delivery of macromolecules before liposomes became popular. To function as carriers, RBC were first loaded up with the macromolecule under hypotonic conditions to induce mild lysis. A fusogen was then used to cause fusion with target cells. Using this technique, thymidine kinase and BSA were introduced into 3T3- 4E mouse cells using Sendai virus as a fusogen (Schlegel & Rechsteiner, 1975). In another study, horseradish peroxidase (HRP) and immunoglobulins (IgG) were delivered into cells expressing hemagglutinin derived from influenza virus by exploiting the fusogenic activity of hemagglutinin under mildly acidic conditions (Doxsey et al., 1985). Fluorescent IgG and

viral integration can trigger the activation of an oncogene or disrupt genes with tumorsuppressive properties. Consequently, several patients became leukemic and at least one has died so far (Sheridan, 2011). More recently, retroviruses were also used to treat two patients diagnosed with X-linked chronic granulomatous disease which affected the ability of their phagocytes to clear bacterial infections (Ott et al., 2006). Insertional mutagenesis was again responsible for a clone of cells whose genes responsible for growth were activated, prompting fears that such cells may turn cancerous. Having said that, viral gene therapy is not expected to be perfect and its success should be assessed relative to existing treatments. Nonetheless, researchers have not completely mastered the use of viruses and there is still a

Diethylaminoethyl (DEAE)-dextran and calcium phosphate (CaPO4) were two of the earliest systems popular in the 1970s and 80s. DEAE-dextran has a cationic polysaccharide backbone and one of the earliest reports in 1965 used this polymer in a 1:1 mixture with nucleic acids to transfect rhesus monkey kidney cells (Vaheri & Pagano, 1965). Authors of this study also insightfully commented that DEAE-dextran, like histones, could bind and protect nucleic acids from degradation. Although these are now fundamental concepts in modern carrier design, it may not have been as obvious in the past. The co-precipitation of CaPO4 with DNA was first described in 1973, when it was observed that Ca2+ (not Mg2+ or Na+) and PO42- ions, at high enough concentrations, could enhance DNA uptake by cells (Graham & Eb, 1973). This technique was further shown to be sensitive to pH, amount of DNA used and even the level of CO2 in the incubator (Chen & Okayama, 1987). Using an optimised protocol, CaPO4-mediated transfection achieved up to 50% efficiency with a murine L cell

Chitosan is a polysaccharide obtained by the deacetylation of chitin, an exoskeletal component of crustaceans. Each deacetylation site contains a primary amine (pKa~6.5) which allows chitosan to bind nucleic acids. The degree of deacetylation also determines its biodegradability and transfection efficiency. Chitosan was first described as a plasmid carrier by Mumper and colleagues in 1995 and is known to be biocompatible, mucoadhesive and virtually non-toxic (MacLaughlin et al., 1998). As such, chitosan has been evaluated in rabbits, although reporter gene expression in that study was low (MacLaughlin et al., 1998). Strong interactions between high molecular chitosan and DNA were blamed for the overlystable complexes that could not release their plasmid cargos. Using lower molecular weight chitosan, *in vitro* transfection efficiency was improved by up to 24 fold (Koping-Hoggard et al., 2004). Adding histidine (pKa~6) residues to chitosan further improved buffering capacity and overall transfection efficiency (Chang et al., 2010). Recently, it was also shown that the introduction of thiol groups in *N,N,N*-trimethylated chitosan improved its efficiency as a

Red blood cells (RBC) were used for the delivery of macromolecules before liposomes became popular. To function as carriers, RBC were first loaded up with the macromolecule under hypotonic conditions to induce mild lysis. A fusogen was then used to cause fusion with target cells. Using this technique, thymidine kinase and BSA were introduced into 3T3- 4E mouse cells using Sendai virus as a fusogen (Schlegel & Rechsteiner, 1975). In another study, horseradish peroxidase (HRP) and immunoglobulins (IgG) were delivered into cells expressing hemagglutinin derived from influenza virus by exploiting the fusogenic activity of hemagglutinin under mildly acidic conditions (Doxsey et al., 1985). Fluorescent IgG and

need for alternative non-viral vectors with safer profiles.

**3.3 Non-viral vectors** 

line (Chen & Okayama, 1987).

siRNA carrier (Varkouhi et al., 2010).

BSA were also delivered into human fibroblast cells using PEG as a fusogen (Wojcieszyn et al., 1981). Dehydration may play a role in PEG-mediated fusion and intriguingly, purification by recrystallisation removed the fusogenic ability of PEG, suggesting that other ingredients in commercial PEG was essential (Wojcieszyn et al., 1983).

Plasmids were initially delivered by synthetic liposomes using concepts adapted from RBC technology. In an early study, plasmids were encapsulated within anionic liposomes and fused with tobacco mesophyll protoplasts using PEG (Deshayes et al., 1985). Since 1987, however, surface binding became popular when liposomes made of *N*-[1-(2,3, dioleyloxy)propyl]-*N*,*N*,*N*-trimethylammonium chloride (DOTMA) were synthesised for the first time (Felgner et al., 1987). DOTMA contains cationic quaternary ammonium groups which can be used for DNA binding. Lipofectin®, the prototypical lipid formulation and a workhorse for transfection experiments today, is a 1:1 mixture of DOTMA and the neutral lipid, dioleoyl phosphatidylethanolamine (DOPE). In this formulation, DOPE functions as a fusogen to aid cellular uptake. Several other formulations of liposomes have been reported. Gao and Huang prepared liposomes made out of DOPE and a novel cationic cholesterol derivative, 3*β*-[*N*-(*N'*,*N'*-dimethylamino)ethane-carbamoyl] cholesterol (DC-chol), and used it to transfect several cell lines with high efficiency (Gao & Huang, 1996). A mixture of *N*′,*N*′ dioctadecyl-*N*-4,8-diaza-10-aminodecanoylglycine amide (DODAG) and DOPE was also proposed to be a novel liposomal formulation that is good for both DNA and siRNA delivery (Mével et al., 2010). In another study, the cationic lipid 1,2-dilinoleyloxy-3 dimethylaminopropane (DLinDMA) was used as a starting template and systematically modified to yield an optimised construct (Semple et al., 2010). Although toxicity remains one of the chief complaints, liposomes are definitely one of the most successful non-viral vectors around. Already, several sustained-release liposomal drug formulations have been approved for human use and thermosensitive liposomes are being evaluated in clinical trials (Hossann et al., 2010). Lipids also dominate in gene therapy trials aimed at using non-viral vectors to treat cystic fibrosis (Griesenbach & Alton, 2009).

An advantage of polymeric carriers is that they offer so much structural diversity that their potential should, theoretically, be limited only by the imagination of chemists. Two of the more archaic examples including PVP and PVA have been mentioned. Poly(D,L-lactic-coglycolic acid) (PLGA), a polymer originally developed for controlled drug release, has also been used to encapsulate plasmids for delivery (Wang et al., 1999). Most polymeric carriers, however, are cationic in nature. Dendritic poly(amido amine) (PAMAM) which contained primary amines for DNA binding was further decorated with PEG and sugars (Wood et al., 2005) or peptide (Wood et al., 2008) for targeting purposes. Linear PAMAM was functionalised with disulfide bonds to make the polymer biodegradable and various chemical side-groups were attached to test for its impact on transfection efficiency (Lin et al., 2007). A large library of poly(β-amino ester)s has been synthesised and systematically screened for transfection efficiency (Green et al., 2006). The effects of molecular weight and charge density of polyphosphoramidates (PPA) on DNA binding and buffering capacity have been reported (Ren et al., 2010). A series of novel enzyme-degradable polycarbonates (PC) with various aliphatic side-chains attached were prepared and shown to effectively transfect cells with virtually no toxicity (Seow & Yang, 2009a). Poly(2-(dimethylamino)ethylmethacrylate) (PDMAEMA) was also shown to transfect cells with high efficiency (Lin et al., 2008). Indeed, there are so many other classes of polymers being developed that it is impossible to do all justice with this paragraph. Nonetheless, amongst all, the most notable polymer must be PEI. PEI was first developed in 1995 (Boussif et al.,

Peptides as Promising Non-Viral Vectors for Gene Therapy 625

Penetratin RQIK IWFQ NRRM KWKK 3rd helix of Antennapedia homeodomain

Tp10 AGYL LGKI NLKA LAAL AKKI L First 6 residues deleted from Tp

CADY GLWR ALWR LLRS LWRL LWRA Peptide carrier PPTG1-derived

HA2 GLFG AIAG FIEN GWEG MIDG Influenza virus hemagglutinin protein INF5 (GLFE AIEG FIEN GWEG nIDG)2K HA2-derived, lysine-connected dimer. n =

The amino acid sequence of PLL and all other peptides subsequently discussed can be found in Table 1. PLL is the first peptide-based vector to be studied intensively. The molecular weight of PLL spans a wide range and depends on the number of repeating units within a chain, or its degree of polymerisation (DP). For the sake of discussion, the molecular weight of PLL is arbitrarily classified as follows: oligolysine (DP<20), low (20≤DP<50), medium

PLL has been successfully employed to deliver a host of different cargos. By covalently coupling human serum albumin or HRP to PLL via amide bonds in a 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide (EDC) catalysed reaction, proteins were delivered into L929 mouse fibroblasts for the first time using such a PLL-mediated strategy (Shen & Ryser, 1978). More recently, streptavidin-conjugated quantum dots (QD) were also attached to biotin-tagged PLL and delivered into HeLa cells (Mok et al., 2008). However, PLL is most frequently used to deliver nucleic acids where the electrostatic attachment is preferred. Although polylysine made out of D-amino acids has been suggested to work better – presumably because they are more resistant to enzymatic degradation (Mitchell et al., 2000) – PLL continues to be favoured in transfection studies. Wu and partner provided one of the earliest examples of PLL being used to condense plasmids for receptor-mediated delivery (Wu & Wu, 1987). In that study, asialoorosomucoid (ASOR) was first covalently attached to PLL with *N*-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as a linker and then used to

SV40 PKKK RKV Simian virus 40 T antigen NLS

Table 1. The amino acid sequences of all the peptides discussed in this chapter.

(50≤DP≤400) or high (DP>400). Most studies use PLL of medium molecular weight.

Galanin + mastoparan (wasp venom)

Membrane disruptive peptide, also a

HSV-1 structural protein

carrier

norleucine

Bee venom

HIV gp41 + SV40 T-antigen

Membrane disruptive peptide

Penetratin analogue

HA2-derived, monomer

Peptide Sequence (single letter code) Short description Poly-L-lysine Kn Frequently, n = 50-400 Tat YGRK KRRQ RRRP PQ HIV-derived, sequence 47-60

Oligoarginine Rn Frequently, n = 7-9

Tp GWTL NSAG YLLG KINL KALA ALAK KIL

VP22 300-residue long sequence given in (Elliott & O'Hare, 1997)

MPG GALF LGFL GAAG STMG AWSQ PKSK RKV

KALA WEAK LAKA LAKA LAKH LAKA LAKA LKAC EA

GALA WEAA LAEA LAEA LAEH LAEA LAEA LEAL AA

EB1 LIRL WSHL IHIW FQNR RLKW

INF7 GLFE AIEG FIEN GWEG MIDG

Melittin GIGA VLKV LTTG LPAL ISWI KRKR QQ

KKK

WYG

**4.1 Poly-L-lysine (PLL)** 

1995) and is frequently used in its branched, high molecular weight (usually 25 kDa) form. Together with liposomes, they are widely acknowledged to be the best non-viral vectors currently available (Putnam, 2006) and frequently serve as standards to which other novel carriers are referenced. A key feature of PEI is that nitrogen (in a mixture of primary, secondary and tertiary amines) accounts for a third of its molecular weight. Since different classes of amines possess characteristic pKa, this ensures that not all the nitrogen will be protonated at a given pH. Furthermore, the proximity of the nitrogen atoms also means that a protonated amine can suppress the protonation of its neighbours due to the energetic penalty that gets incurred by situating like charges adjacent to one another (Suh et al., 1994). This blend of protonated and yet-to-be protonated amines is then suggested to endow PEI with its strong DNA binding and buffering abilities. The intracellular trafficking properties of PEI/DNA complexes has been studied (Godbey et al., 1999) and targeting moieties such as mannose (Diebold et al., 1999) and transferrin (Kircheis et al., 1997) were also coupled onto PEI. However, two of the main problems of PEI are its toxicity and nonbiodegradability. In response to the latter, PEI was functionalised with reducible disulfide bonds (Lee et al., 2007) or hydrolysable ester bonds (Liu et al., 2008) to facilitate biodegradation and at the same time, to aid its intracellular plasmid release.
