**3. Extracellular barriers to overcome. Non-invasive routes of administration**

Depending on the administration route and the target organ, gene delivery systems must overcome several extracellular barriers *in vivo* before reaching the target cells. As mentioned earlier, cancer diseases represent 60% of all clinical trials in gene therapy; yet other pathologies such as infectious, neurodegenerative, ocular and pulmonary diseases merit special attention, which, in sum, represent 10% of clinical trials in gene therapy. The principal and most studied route of administration of non-viral gene delivery systems for those diseases is the intravenous administration. Here, vectors need to be properly designed in order to overcome all the hurdles this route presents. Moreover, when specific tissues need to be targeted, such as the brain, the eye or the lungs, additional extracellular barriers appear and vectors have to be able to surpass them too. In this section, we will review the principal systemic barriers following the intrave‐ nous administration of non-viral gene delivery systems, as well as the additional tissue-specific barriers DNA/vector complexes have to overcome. We will also describe several attempts that have been made in order to overcome those barriers using invasive and alternative noninvasive routes of administration. Many efforts are being conducted to achieve effective strategies for safe non-viral gene delivery platforms based on non-invasive administration routes.

#### **3.1. Intravenous administration**

[40]. In addition, highly basic polymers such as polylysine and protamine, the highly basic sequence of which resembles typical NLS sequences, have been used as potential agents to

**Figure 5.** Cell entry pathways and intracellular trafficking of non-viral gene delivery systems. A) Phagocytosis. B) Cla‐ thrin-mediated endocytosis (CME). C) Caveolae-mediated endocytosis (CvME). D) Macropinocytosis. Internalized vec‐ tor/DNA complexes following the CME pathway escape from endosomes and can be transported into the nucleus by the importins if they contain an NLS. When vector and DNA dissociate from each other, the transgene is expressed in the target cells. Yellow filaments represent actin; red chain represents clathrin coat; green filaments represent caveolin dimers; orange little circles represent the small GTPase dynein; blue filaments represent opsonins. (Adapted from [23;

Finally, it should be considered that, once inside the nucleus, the non-viral vector itself may constitute a barrier to transgene expression. In fact, the agent used to condense the DNA could potentially interfere with the access of the cellular transcription machinery to the transgene promoter, thereby reducing or preventing its expression [40]. Still, premature release of DNA from the vector may expose the DNA to enzymatic degradation before expression can occur [40,44]. For liposomal-based vectors, DNA displacement from the vector seems to be connected to endosomal escape, driven by the anionic lipids of the endosomal membrane that neutralize the charge of the cationic lipids in the liposomal formulation [40,45,46]. In contrast, polycation/ DNA complexes appear to release from each other in the nucleus through exchange of the polycations in the complexes with the protein components of the surrounding proteins

enhance nuclear targeting when complexed with DNA [40].

158 Gene Therapy - Principles and Challenges

24])

For many cancer forms, and specially disseminated cancer diseases, treatment needs to be administered systemically. Thus, intravenously administered current gene delivery systems to treat cancer should be able to transport and deliver the genetic cargo into cancerous cells.

The principal challenge of systemically administered DNA is to resist the extracellular enzymatic degradation, since DNA is subject to enzymatic degradation from the point of entry. However, it is possible to considerably surmount this hurdle by condensing the negatively charged DNA with the positively charged non-viral vectors [13].

Secondly, the other major extracellular barrier in the systemic route is the non-specific binding of the non-viral vector/DNA complex, which has a net positive charge, with blood cells and serum proteins such as albumin, complements, immunoglobulins and fibronectin,which have a negative surface charge [49]. These interactions could potentially end in aggregation or dissociation of vector/DNA complexes, resulting in their rapid clearance and elimination by the reticuloendothelial systems [49].

A third obstacle in systemic delivery might be the colloidal instability of non-viral vector/DNA complex formulations in the extracellular environment, which can also result in the aggrega‐ tion of the complexes [49]. Fourth, vascular system is an extracellular barrier to be considered since it limits the size of the nanoparticles that can pass through the endothelial cells, which are relatively small and have tight junctions [49].

Finally, the activation of the immune system by the foreign vector/DNA complexes is an extracellular issue to be taken into consideration as well. In fact, foreign synthetic vectors can also induce an inflammatory response and/or complement activation, and hydrophobic particles can be eliminated by mononuclear phagocytic system through opsonization [49].

In order to overcome all these systemic barriers, non-viral vectors should be structurally modified. Formulation of gene delivery vectors is a key factor in determining their bioavaila‐ bility and transfection efficiency *in vivo* [49]. Some of the strategies that are being developed to improve the properties of nanoparticles in the extracellular environment are discussed below.

The most employed strategy to increase the stability of vector/DNA complexes is shielding the outer surface of complexes with poly(ethylene glycol) or PEG [50]. Because of its highly hydrophobic nature, PEG produces a steric barrier against nuclease degradation and aggre‐ gation of nanoparticles in blood circulation [49]. However, despite the promising results, some difficulties exist in conjugation of PEG to gene delivery systems. PEGylation could decrease binding ability of non-viral vectors to DNA causing instability of lipo- or polyplexes in blood circulation. It may also affect the binding of vector/DNA complexes to receptors on the cell membrane [51]. Moreover, PEGylation can induce accelerated blood clearance due to activa‐ tion of splenic synthesis of anti-PEG IgM antibody after first injection, resulting in the opsonization of the subsequent doses [49]. The length and the degree of PEGylation can also affect the ability of DNA condensation and biodistribution of gene carriers *in vivo*, and the optimal PEG length and content depends on gene carrier systems [52].

Consequently, considerable research has been made with the aim of compensating the negative effect of PEGylation in non-viral gene delivery systems. As reported in a recent study, one possible solution is to replace PEG by some hydrophilic polymers such as poly(N-vinyl-2 pyrrolidone) (PVP), poly(4-acryloylmorpholine), or poly(N,N-dimethylacrylamide) [53]. Coating of nanoparticles with these polymers led to extended residence of the nanoparticles in blood circulation in rats, although they had a shorter half-life than the PEG-coated nano‐ particles [53]. Other strategies include providing stability against serum compounds and enzymatic digestion using copolymers of poly(L-lysine) and poly(2-methyl-2-oxazoline) [54], the pH-sensitive shielding of DNA polyplexes or lipoplexes (e.g. with PEG-acetal-MAL or maleimide moiety) [55], the use of enzymatically cleavable PEG linkers (e.g. PEG-peptide-DOPE or PPD that is cleaved in a matrix metalloproteinase-rich environment) [56], or the production of reducible PEG nanoparticles (e.g. PEG and chitosan bound through disulphide bridges) [57].

Besides PEGylation, other chemical and structural modifications can be applied to gene delivery agents in order to overcome the systemic barriers. In the case of cationic lipid-based non-viral vectors, incorporation of cholesterol can stabilize lipoplexes against binding to red blood cells [49]. In the case of cationic polymer-based non-viral vectors, conjugation of lactose to chitosan polyplexes has shown excellent DNA binding ability, good protection of DNA from nuclease, and the suppression of self-aggregation and serum-induced aggregation [58]. Therefore, current research has focused on multifunctional and diverse non-viral gene carriers that can be adjusted for each particular condition.

#### **3.2. Targeting specific tissues: additional extracellular barriers**

When targeting specific tissues, vectors have to be able to surpass additional barriers as well. Here, we will focus on specific extracellular barriers present in gene delivery to the central nervous system (CNS), to the eye and to the lungs. The invasive and alternative non-invasive routes of administration that avoid those barriers will also be discussed.

#### *3.2.1. Gene delivery to CNS*

The principal challenge of systemically administered DNA is to resist the extracellular enzymatic degradation, since DNA is subject to enzymatic degradation from the point of entry. However, it is possible to considerably surmount this hurdle by condensing the negatively

Secondly, the other major extracellular barrier in the systemic route is the non-specific binding of the non-viral vector/DNA complex, which has a net positive charge, with blood cells and serum proteins such as albumin, complements, immunoglobulins and fibronectin,which have a negative surface charge [49]. These interactions could potentially end in aggregation or dissociation of vector/DNA complexes, resulting in their rapid clearance and elimination by

A third obstacle in systemic delivery might be the colloidal instability of non-viral vector/DNA complex formulations in the extracellular environment, which can also result in the aggrega‐ tion of the complexes [49]. Fourth, vascular system is an extracellular barrier to be considered since it limits the size of the nanoparticles that can pass through the endothelial cells, which

Finally, the activation of the immune system by the foreign vector/DNA complexes is an extracellular issue to be taken into consideration as well. In fact, foreign synthetic vectors can also induce an inflammatory response and/or complement activation, and hydrophobic particles can be eliminated by mononuclear phagocytic system through opsonization [49]. In order to overcome all these systemic barriers, non-viral vectors should be structurally modified. Formulation of gene delivery vectors is a key factor in determining their bioavaila‐ bility and transfection efficiency *in vivo* [49]. Some of the strategies that are being developed to improve the properties of nanoparticles in the extracellular environment are discussed

The most employed strategy to increase the stability of vector/DNA complexes is shielding the outer surface of complexes with poly(ethylene glycol) or PEG [50]. Because of its highly hydrophobic nature, PEG produces a steric barrier against nuclease degradation and aggre‐ gation of nanoparticles in blood circulation [49]. However, despite the promising results, some difficulties exist in conjugation of PEG to gene delivery systems. PEGylation could decrease binding ability of non-viral vectors to DNA causing instability of lipo- or polyplexes in blood circulation. It may also affect the binding of vector/DNA complexes to receptors on the cell membrane [51]. Moreover, PEGylation can induce accelerated blood clearance due to activa‐ tion of splenic synthesis of anti-PEG IgM antibody after first injection, resulting in the opsonization of the subsequent doses [49]. The length and the degree of PEGylation can also affect the ability of DNA condensation and biodistribution of gene carriers *in vivo*, and the

Consequently, considerable research has been made with the aim of compensating the negative effect of PEGylation in non-viral gene delivery systems. As reported in a recent study, one possible solution is to replace PEG by some hydrophilic polymers such as poly(N-vinyl-2 pyrrolidone) (PVP), poly(4-acryloylmorpholine), or poly(N,N-dimethylacrylamide) [53]. Coating of nanoparticles with these polymers led to extended residence of the nanoparticles

optimal PEG length and content depends on gene carrier systems [52].

charged DNA with the positively charged non-viral vectors [13].

the reticuloendothelial systems [49].

160 Gene Therapy - Principles and Challenges

below.

are relatively small and have tight junctions [49].

The CNS possesses particular anatomical and physiological properties that make gene delivery to CNS specially challenging. The CNS is protected by the blood-brain-barrier (BBB), which consists of tightly joined capillary endothelial cells [19], and it is considered to be impermeable for almost 100% of the macromolecular drugs and over 98% of small molecule drugs [59]. The spinal cord is part of the CNS and it is protected by the blood-cerebrospinal fluid barrier (BCSFB), which is constituted of choroid plexus epithelial cells and restricts the free diffusion of molecules into the cerebrospinal fluid (CSF) [19]. Transport into the CNS of essential nutrients, such as glucose and amino acids, occurs through specific receptors present in the BBB and the BSCFB [19]. Within the CNS, distinct cell types exist including neurons and different types of glial cells; neurons are particularly challenging to transfect and it is thought this is attributable to their post-mitotic nature, their complex structure and the complexity of neuronal networking [19].

Most of the strategies to cross the BBB upon systemic administration of non-viral vector/DNA complexes exploit receptor-mediated uptake of molecules such as transferrin (Tf), lactoferrin and insulin, since receptors of those molecules are expressed on many cell types, including neurons and the capillary endothelial cells of the BBB [19]. By attaching a ligand for those receptors to the non-viral delivery system, one can enhance the transport of the vector/DNA complex towards the CNS. Another strategy known as 'Molecular Trojan Horse' uses pepti‐ domimetic monoclonal antibodies that are designed to target specific receptors on the BBB and induce receptor-mediated transcytosis of the non-viral delivery system into the CNS [60]. Other approaches investigated for CNS delivery of conventional pharmaceutics upon systemic administration include transient mechanical disruption of the BBB and RNAi-mediated knockdown of tight junction proteins [19].

The ultimate goal for CNS gene therapeutics is delivery by systemic route, which is the most acceptable for clinical use. Nevertheless, in view of the high amount of extracellular barriers that vectors must overcome, many studies have attempted different routes of administration. To date, several pre-clinical studies have essayed local administration to the brain, either by injection or by infusion. However, even if local administration to the brain eludes the extrac‐ ellular barriers to access the CNS, the need for brain surgery to infuse a gene therapy vector clearly limits the clinical applicability for this approach.

Intranasal delivery offers a novel and non-invasive means by which non-viral gene delivery systems can gain access to the brain. The mechanisms by which intranasally delivered substances enter the CNS have not been fully elucidated, but an accumulating amount of evidence suggests that substances can reach the brain through a combination of perineuronal, perivascular and lymphatic transport pathways. In addition, the prevailing nose-to-brain pathway will largely depend on the region where the delivered agent is placed within the nasal cavity and the physicochemical properties of the therapeutic being administered [61]. It is currently accepted that the intranasally administered substance can reach the CNS by three main pathways: (i) direct paracellular or transcellular transport via the olfactory neurons or olfactory epithelial cells ('olfactory neural pathway'), (ii) transport via the trigeminal nerves ('trigeminal pathway') or (iii) indirectly, *via* blood vasculature and/or lymphatic system ('systemic pathway') [62]. The nasal mucosa is highly vascularized, and the blood vessels allow passage of drugs following nasal administration in nano-drug delivery systems; however, the substance that has been absorbed into the systemic circulation has to cross the BBB in order to reach the CNS [61]. Following olfactory and trigeminal nerve pathways, drug is delivered to the olfactory bulbs and to more caudal brain areas, respectively [63]. Within the brain, pulsatile flow in perivascular spaces has been postulated to allow for widespread transport of molecules within interstitial fluid to sites deep in parenchyma [63,64].

Advantages of intranasal administration include ease of administration (non-invasive), rapid dose absorption via highly vascularized mucosa, large nasal mucosa surface area for dose absorption, avoidance of the gastrointestinal tract, first-pass metabolism and fewer side effects, among others [65]. Moreover, intranasal administration confers improved convenience and compliance compared to other more invasive routes and it allows self-administration [65]. Disadvantages of this route include that nasal congestion could interfere with dose absorption, that the amount of dose that reaches the CNS varies with each agent and that the frequent use of this route leads to mucosal damage [65]. In addition, the administered formulation can undergo rapid clearance from nasal cavity by the mucociliary system [59,66]. This latter drawback can be overcome by adding a mucoadhesive substance to the formulations. For nonviral-vector-based gene delivery systems, chitosan is an attractive excipient that can confer both bio-adhesion and absorption properties, and it is the most widely investigated absorption enhancer material both in terms of efficiency and safety [63]. Chitosan is able to interact through its positively charged amino groups with the anionic counterpart present in the mucus layers, mainly sialic acid, and to affect permeability of the epithelial membrane by the transient opening of the tight junctions in the epithelial cells [67]. In a recent study, another substance, the non-ionic surfactant laureate sucrose ester has been also reported to be an effective intranasal absorption enhancer [68].

Recently, the first report that intranasal delivery of DNA nanoparticles can bypass the BBB and transfect and express the encoded protein in rat brain has been published, thereby affording a non-invasive approach for gene therapy in CNS disorders [63]. Authors demon‐ strated that intranasal delivery of unimolecularly compacted DNA nanoparticles, which consist of single molecules of plasmid DNA encoding enhanced green fluorescent protein (eGFP) compacted with 10 kDa commercial peptide (PEG-substituted lysine 30 mers or CK30PEG10k), successfully transfects cells and leads to the expression of the eGFP in the rat brain [63]. The results further suggest that the cells transfected within the brain are likely to be pericytes, and that the distribution of nasally administered substances occurs via perivas‐ cular transport [63]. Additionally, another recent study has reported brain (cortex and hippocampus) transfection upon intranasal administration of chitosan and polyethyleneimine (PEI)-coated magnetic micelles [69]. Even if those nanoparticles were able to reach the brain presumably because of a transient disruption of the BBB following mild traumatic brain injury, the results show that the intranasal route might be useful for targeting the brain. Although further optimization of the dose, dosing regimen and dose interval is needed to achieve appropriate levels of transgene expression [63], the promising results will certainly encourage the research in the field of intranasal administration of non-viral-vector-based gene delivery systems, which has clinical importance due to its non-invasive nature.

#### *3.2.2. Gene delivery to the eye*

complex towards the CNS. Another strategy known as 'Molecular Trojan Horse' uses pepti‐ domimetic monoclonal antibodies that are designed to target specific receptors on the BBB and induce receptor-mediated transcytosis of the non-viral delivery system into the CNS [60]. Other approaches investigated for CNS delivery of conventional pharmaceutics upon systemic administration include transient mechanical disruption of the BBB and RNAi-mediated

The ultimate goal for CNS gene therapeutics is delivery by systemic route, which is the most acceptable for clinical use. Nevertheless, in view of the high amount of extracellular barriers that vectors must overcome, many studies have attempted different routes of administration. To date, several pre-clinical studies have essayed local administration to the brain, either by injection or by infusion. However, even if local administration to the brain eludes the extrac‐ ellular barriers to access the CNS, the need for brain surgery to infuse a gene therapy vector

Intranasal delivery offers a novel and non-invasive means by which non-viral gene delivery systems can gain access to the brain. The mechanisms by which intranasally delivered substances enter the CNS have not been fully elucidated, but an accumulating amount of evidence suggests that substances can reach the brain through a combination of perineuronal, perivascular and lymphatic transport pathways. In addition, the prevailing nose-to-brain pathway will largely depend on the region where the delivered agent is placed within the nasal cavity and the physicochemical properties of the therapeutic being administered [61]. It is currently accepted that the intranasally administered substance can reach the CNS by three main pathways: (i) direct paracellular or transcellular transport via the olfactory neurons or olfactory epithelial cells ('olfactory neural pathway'), (ii) transport via the trigeminal nerves ('trigeminal pathway') or (iii) indirectly, *via* blood vasculature and/or lymphatic system ('systemic pathway') [62]. The nasal mucosa is highly vascularized, and the blood vessels allow passage of drugs following nasal administration in nano-drug delivery systems; however, the substance that has been absorbed into the systemic circulation has to cross the BBB in order to reach the CNS [61]. Following olfactory and trigeminal nerve pathways, drug is delivered to the olfactory bulbs and to more caudal brain areas, respectively [63]. Within the brain, pulsatile flow in perivascular spaces has been postulated to allow for widespread transport of molecules

Advantages of intranasal administration include ease of administration (non-invasive), rapid dose absorption via highly vascularized mucosa, large nasal mucosa surface area for dose absorption, avoidance of the gastrointestinal tract, first-pass metabolism and fewer side effects, among others [65]. Moreover, intranasal administration confers improved convenience and compliance compared to other more invasive routes and it allows self-administration [65]. Disadvantages of this route include that nasal congestion could interfere with dose absorption, that the amount of dose that reaches the CNS varies with each agent and that the frequent use of this route leads to mucosal damage [65]. In addition, the administered formulation can undergo rapid clearance from nasal cavity by the mucociliary system [59,66]. This latter drawback can be overcome by adding a mucoadhesive substance to the formulations. For nonviral-vector-based gene delivery systems, chitosan is an attractive excipient that can confer

knockdown of tight junction proteins [19].

162 Gene Therapy - Principles and Challenges

clearly limits the clinical applicability for this approach.

within interstitial fluid to sites deep in parenchyma [63,64].

The eye is an attractive target organ for gene therapy because of its unique characteristics. The tissue volume to be treated is small, the therapeutic concentration to be administered is relatively low and the diffusion of active products from the eye to the circulation is minimal [70]. In addition, the eye benefits from a relative immune privilege, minimizing the potential immune and inflammatory reactions that may follow the intraocular injections of foreign agents [70].

In general, gene delivery systems for eye diseases range from simple eye drops and oint‐ ments to more advanced bio- and nanotechnology-based systems such as muco-adhesive systems, polymers, liposomes and ocular inserts. Most of these technologies were developed for front-of-the-eye ophthalmic therapies and are not applicable as back-of-the-eye delivery systems [71].

When the systemic administration is used to target the eye, non-viral vector/DNA complexes must cross the blood-ocular-barrier (BOB) to reach the ocular tissue. This constitutes a real challenge, since the BOB is composed of tight epithelial junctions. Two principal strategies to overcome this barrier are the use of vectors smaller than 100 nm to allow intracellular passage across the BOB [72] and the use of ligand-equipped vectors that recognize specific receptors in the BOB [73]. Therefore, even if the intravenous route permits the delivery of larger volumes of the formulations as well as repeated administrations, the therapeutic effect achieved by this method is often limited by the factors restricting the access to the eye.

Invasive methods such as intravitreal injection, subconjuntival injection and subretinal injection can bypass some of those barriers, and intravitreal and subretinal injections are currently considered as the most effective and common methods of gene delivery to retinal ganglion cells and to inner layers of the retina, respectively. However, these methods are very invasive and repeated gene delivery to the eye using such methods can cause further damage of the eye like retinal detachment, haemorrhages, and sub- or pre-retinal fibrosis [71]. There‐ fore, non-invasive and effective methods for ocular gene delivery are needed. In this regard, topical administration in the form of eye drops is a non-invasive delivery method that can be performed repeatedly with minimal side effects [71].

However, the non-invasive route of administration is perhaps the most ambitious goal because the barriers associated with topical gene delivery to the posterior ocular tissue are the most challenging. First, vector/DNA complexes have to surpass the tear film, which is an aqueous layer covered by lipids and underlined by mucin that covers the corneal and conjuctival layers [71]. This tear film restricts the bioavailability of applied formulations because of the tear turnover rate and the lacrimal and nasolacrimal drainage [85]. Strategies to overcome this barrier include addition of viscosity enhancers such as cellulose derivatives or thermorever‐ sible poloxamer gels [71] and, most importantly, the incorporation of muco-adhesive polymers such as chitosan and hyaluronic acid derivatives in gene delivery systems [74]. Second, ocular tissue barriers such as the cornea, conjunctiva, sclera and choroid, contain epithelial tight junctions, proteoglycan matrices and fibril collagen networks within their structures, which contribute to restrict the passage of vector/DNA complexes to the neuroretina [71]. Finally, the vitreous is an aqueous biogel composed of collagen, hyaluronan and proteoglycans that hinders transfection of the retinal cells [71]. Strategies to overcome those barriers include all the above-mentioned methods from the use of vectors of suitable dimensions to the use of specific ligands and muco-adhesive polymers [71].

The majority of success in ocular gene therapy research thus far was accomplished for applications involving the anterior part of the eye, using mainly viral-based delivery systems and invasive delivery methods. Interestingly, effective gene delivery to the retina and retinal pigment epithelium using non-viral vectors has been recently reported; however, in this study magnetic nanoparticles were administered invasively, through intravitreal and subretinal injections [75]. Rather than in gene delivery, significant advances have been made in drug delivery systems to target the posterior part of the eye using non-invasive administration routes. For instance, in a recent study, it was reported that surface-modified submicron-sized lipid emulsions could be promising vehicles of hydrophobic drug delivery to the ocular posterior segment [76]. In that study, researchers performed surface modification of the lipid emulsions using a positive charge inducer and the functional polymers chitosan and polox‐ amer 407. Authors suggested that poloxamer 407 increased the lipid emulsion retention time on the eye surface by its adhesive properties, therefore enhancing gene delivery to the ocular posterior segment. Additionally, another study has reported successful drug delivery to the posterior segment of the eye of rats and rabbits using annexin A5-associated liposomes [77]. Here, authors suggested that annexin A5 mediated endocytosis can enhance the delivery of associated lipidic drug delivery vehicles across biological barriers. Moreover, a novel study has reported the topical drug delivery to retinal pigment epithelium with microfluidizer produced small liposomes, which might be an attractive option for drug delivery to the posterior segment tissues of the eye [78]. It may be reasonable that some of the advances in drug delivery will be applicable for gene delivery systems as well, and they will probably inspire further strategies for non-invasive, non-viral gene therapy platforms aimed at targeting the posterior segment of the eye.

#### *3.2.3. Gene delivery to the lungs*

across the BOB [72] and the use of ligand-equipped vectors that recognize specific receptors in the BOB [73]. Therefore, even if the intravenous route permits the delivery of larger volumes of the formulations as well as repeated administrations, the therapeutic effect achieved by this

Invasive methods such as intravitreal injection, subconjuntival injection and subretinal injection can bypass some of those barriers, and intravitreal and subretinal injections are currently considered as the most effective and common methods of gene delivery to retinal ganglion cells and to inner layers of the retina, respectively. However, these methods are very invasive and repeated gene delivery to the eye using such methods can cause further damage of the eye like retinal detachment, haemorrhages, and sub- or pre-retinal fibrosis [71]. There‐ fore, non-invasive and effective methods for ocular gene delivery are needed. In this regard, topical administration in the form of eye drops is a non-invasive delivery method that can be

However, the non-invasive route of administration is perhaps the most ambitious goal because the barriers associated with topical gene delivery to the posterior ocular tissue are the most challenging. First, vector/DNA complexes have to surpass the tear film, which is an aqueous layer covered by lipids and underlined by mucin that covers the corneal and conjuctival layers [71]. This tear film restricts the bioavailability of applied formulations because of the tear turnover rate and the lacrimal and nasolacrimal drainage [85]. Strategies to overcome this barrier include addition of viscosity enhancers such as cellulose derivatives or thermorever‐ sible poloxamer gels [71] and, most importantly, the incorporation of muco-adhesive polymers such as chitosan and hyaluronic acid derivatives in gene delivery systems [74]. Second, ocular tissue barriers such as the cornea, conjunctiva, sclera and choroid, contain epithelial tight junctions, proteoglycan matrices and fibril collagen networks within their structures, which contribute to restrict the passage of vector/DNA complexes to the neuroretina [71]. Finally, the vitreous is an aqueous biogel composed of collagen, hyaluronan and proteoglycans that hinders transfection of the retinal cells [71]. Strategies to overcome those barriers include all the above-mentioned methods from the use of vectors of suitable dimensions to the use of

The majority of success in ocular gene therapy research thus far was accomplished for applications involving the anterior part of the eye, using mainly viral-based delivery systems and invasive delivery methods. Interestingly, effective gene delivery to the retina and retinal pigment epithelium using non-viral vectors has been recently reported; however, in this study magnetic nanoparticles were administered invasively, through intravitreal and subretinal injections [75]. Rather than in gene delivery, significant advances have been made in drug delivery systems to target the posterior part of the eye using non-invasive administration routes. For instance, in a recent study, it was reported that surface-modified submicron-sized lipid emulsions could be promising vehicles of hydrophobic drug delivery to the ocular posterior segment [76]. In that study, researchers performed surface modification of the lipid emulsions using a positive charge inducer and the functional polymers chitosan and polox‐ amer 407. Authors suggested that poloxamer 407 increased the lipid emulsion retention time on the eye surface by its adhesive properties, therefore enhancing gene delivery to the ocular

method is often limited by the factors restricting the access to the eye.

performed repeatedly with minimal side effects [71].

164 Gene Therapy - Principles and Challenges

specific ligands and muco-adhesive polymers [71].

Pulmonary gene therapy is considered for the treatment of a variety of lung diseases like cystic fibrosis, asthma, emphysema and lung cancer [79]. Depending on the respiratory disease to be treated, the target cells in the lung can vary from epithelial cells, alveolar cells, macrophages, respiratory stem cells or endothelial cells [79]. Besides, the nucleic acid cargo needs to be delivered to cells in the target region of the lung. Nevertheless, this is severely limited by the pulmonary architecture, the presence of mucus, the clearance mechanisms and the activation of the immune system [79]. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are techniques that can be used to administer materials of interest to the lungs. Considering the interest of non-invasive administration routes for clinical applica‐ tions, aerosolized non-viral vector/DNA complexes for inhalation would be the ideal choice for lung gene therapy. There are many advantages to administering medications to the lungs as an aerosol, such as the high local concentration by delivery directly to the airways, and the pain- and needle-free delivery.

Respiratory secretions, which include mucus and alveolar fluid, are the most important extracellular barriers for lung gene delivery. Respiratory mucus is one of the most important defence mechanisms and it is mainly composed by a three-dimensional network of crosslinked mucin chains, which gives the mucus viscoelastic properties [79]. The major proteins in respiratory mucus are albumin, proteases, anti-proteases, immunoglobulins, lysozyme and lactoferrin, and the respiratory secretions of patients with cystic fibrosis or respiratory infections also contain huge amounts of DNA and actin [79]. The alveolar fluid is a thin continuous layer of pulmonary surfactant that covers the alveolar epithelium and it comprises phospholipids and specific surfactant-associated proteins [79].

Respiratory mucus can act as a barrier towards pulmonary gene delivery in several ways. The biopolymer network of mucus limits the diffusion of complexes by sterical obstruction or by binding the complexes [79]. Also, negatively charged and non-cross-linked macromolecules of mucus, as well as other components present in the mucus such as antibodies, can bind to the surface of vector/DNA complexes. These interactions may cause: (i) entrapment of the vector/DNA complexes in the mucus, (ii) aggregation of the complexes due to neutralization of their surface charges, (iii) release of the DNA cargo from the vector, and (iv) an inefficient cell binding of the complex due to shielding of their positive charges or their receptor binding ligands [79]. Finally, the mucus blanket is continuously removed via mucociliary transport or coughing. Therefore, the vector/DNA complex should be able to cross the mucus before they are cleared from the respiratory tract. The diffusion coefficient of the complexes in the mucus, the thickness of the mucus layer and the rate of mucus clearance will determine whether the vector/DNA complexes will reach the epithelial cells [79].

Regarding the alveolar fluid as a barrier towards pulmonary gene therapy, the presence of this surfactant layer can inhibit transfection of cationic lipid-based vector/DNA complexes [79]. It has been suggested that this inhibitory effect results from disintegration of the lipoplexes by the negatively charged lipids present in the surfactant layer, leading to accessibility of nucleases to the DNA cargo and thus its degradation and loss of function [80]. On the other hand, non-viral vectors based on cationic polymers such as PEI might be more resistant to detrimental effects by pulmonary surfactant [79].

Several strategies have been developed in order to overcome the extracellular barriers of lung gene delivery. Size and surface properties of non-viral vector/DNA complexes have a pivotal role in determining their behaviour in respiratory secretions. Most efforts have been conducted to increase the mobility of vector/DNA complexes in respiratory mucus and to avoid interac‐ tions of complexes with respiratory secretions.

There are different methods to increase non-viral vector/DNA complex mobility through the respiratory mucus. One straightforward mechanism consists of adding mucolytic agents that hydrolyze mucins present in the mucus [79]. Also, it has been demonstrated that N-acetylcys‐ teine and its derivatives lower the viscosity and elasticity of mucus by reducing the disulphide bridges between the subunits of mucins [81]. As future directions, research is focusing on functionalized nanoparticles with mucolytic agents able to cut a way through the mucus, enhancing their transport across the extracellular matrix [79].

On the other hand, the principal strategy to avoid interactions between vector/DNA complexes and components of biological fluids is the shielding of the complexes by modification with biocompatible hydrophilic but biologically inert polymers [79]. Shielding of vector/DNA complexes may not only be important to reduce interaction with mucus and alveolar fluid components but also diminish clearance by alveolar macrophages. For instance, shielding the positive surface charges of vector/DNA complexes with neutral hydrophilic polymers such as polyethylene glycol (PEG) favours their physicochemical stability and their gene transfer capacity [79].

A number of challenges must be overcome before pulmonary gene therapy becomes a reality, such as the development of gene vectors that can more efficiently penetrate the mucus barrier [82]. However, real advances have been made in recent years, novel aerosol therapeutic modalities are currently being investigated for lung cancer, and inhaled gene therapy has already presented safety and effectiveness in cystic fibrosis [83].

As summarized in Figure 6, non-viral vector/DNA complexes have to overcome several extracellular barriers before binding the target cells and initiating the intracellular trafficking towards the nucleus, where transgene expression will occur. Intravenous injection is the most widely used administration route but it presents many hurdles that hamper effective gene delivery. Local administration routes are being explored in order to directly target the tissue of interest and avoid systemic barriers, but they often implicate invasive procedures (e.g. brain surgery). Many efforts are currently focused on the study of non-invasive routes of adminis‐ tration that can equally avoid the systemic barriers (e.g. intranasal administration). Besides, local administration into specific tissues such as the eye or the lungs, involve additional barriers that gene delivery systems have to elude. Surface modifications of the vector/DNA formulations are the most employed strategies to overcome both systemic and tissue-specific barriers. It is expected that future non-viral gene delivery systems will be based on multifunc‐ tional vectors and that they will allow a non-invasive administration of therapeutic genes into target tissues.

ligands [79]. Finally, the mucus blanket is continuously removed via mucociliary transport or coughing. Therefore, the vector/DNA complex should be able to cross the mucus before they are cleared from the respiratory tract. The diffusion coefficient of the complexes in the mucus, the thickness of the mucus layer and the rate of mucus clearance will determine whether the

Regarding the alveolar fluid as a barrier towards pulmonary gene therapy, the presence of this surfactant layer can inhibit transfection of cationic lipid-based vector/DNA complexes [79]. It has been suggested that this inhibitory effect results from disintegration of the lipoplexes by the negatively charged lipids present in the surfactant layer, leading to accessibility of nucleases to the DNA cargo and thus its degradation and loss of function [80]. On the other hand, non-viral vectors based on cationic polymers such as PEI might be more resistant to

Several strategies have been developed in order to overcome the extracellular barriers of lung gene delivery. Size and surface properties of non-viral vector/DNA complexes have a pivotal role in determining their behaviour in respiratory secretions. Most efforts have been conducted to increase the mobility of vector/DNA complexes in respiratory mucus and to avoid interac‐

There are different methods to increase non-viral vector/DNA complex mobility through the respiratory mucus. One straightforward mechanism consists of adding mucolytic agents that hydrolyze mucins present in the mucus [79]. Also, it has been demonstrated that N-acetylcys‐ teine and its derivatives lower the viscosity and elasticity of mucus by reducing the disulphide bridges between the subunits of mucins [81]. As future directions, research is focusing on functionalized nanoparticles with mucolytic agents able to cut a way through the mucus,

On the other hand, the principal strategy to avoid interactions between vector/DNA complexes and components of biological fluids is the shielding of the complexes by modification with biocompatible hydrophilic but biologically inert polymers [79]. Shielding of vector/DNA complexes may not only be important to reduce interaction with mucus and alveolar fluid components but also diminish clearance by alveolar macrophages. For instance, shielding the positive surface charges of vector/DNA complexes with neutral hydrophilic polymers such as polyethylene glycol (PEG) favours their physicochemical stability and their gene transfer

A number of challenges must be overcome before pulmonary gene therapy becomes a reality, such as the development of gene vectors that can more efficiently penetrate the mucus barrier [82]. However, real advances have been made in recent years, novel aerosol therapeutic modalities are currently being investigated for lung cancer, and inhaled gene therapy has

As summarized in Figure 6, non-viral vector/DNA complexes have to overcome several extracellular barriers before binding the target cells and initiating the intracellular trafficking towards the nucleus, where transgene expression will occur. Intravenous injection is the most widely used administration route but it presents many hurdles that hamper effective gene

vector/DNA complexes will reach the epithelial cells [79].

166 Gene Therapy - Principles and Challenges

detrimental effects by pulmonary surfactant [79].

tions of complexes with respiratory secretions.

capacity [79].

enhancing their transport across the extracellular matrix [79].

already presented safety and effectiveness in cystic fibrosis [83].

**Figure 6.** Overview of the systemic and tissue-specific (SNC, eye, lungs) extracellular barriers in non-viral gene therapy.
