**4. Biological barriers**

1999 and 2007, six clinical trials were conducted with these kinds of vectors in CF [8]. Phase I clinical trials demonstrated that a single-dose administration of AAV in the respiratory tract of patients affected by CF was safe and well tolerated [36]. Nevertheless, subsequent studies, with repeated doses in more patients, did not report significant improvement in lung function [37]. This lack of efficacy was mainly attributed to the low DNA loading capacity of AAV, which prevented loading the 4.7 kb of the CFTR gene [8]. In addition, AAV capsid-specific immune responses limited repeated administrations in patients [8]. Nowadays, some interesting strategies are being developed in order to minimize adaptive immune responses after repeated administration, such as the design of hybrid AAV capsids or the removal of CpG

Lentiviruses have an integrative nature and have shown long-term and stable transgene expression when administered in the respiratory tract of mice, which minimizes the need for repeated administration [40]. Additionally, the packaging of full-length CFTR gene and promoters is not limited by size. Therefore, nowadays they are considered promising vectors for the treatment of CF [41]. However, in order to consider its use in clinical practice, some concerns still need to be resolved, such as the scaling in the production of these vectors and the control of the place where the transgene is inserted into the genome of the pulmonary cells, which could increase the tumorigenicity potential of such viral vectors due to random integration [20]. Consequently, such viral vectors could be more suitable for ex vivo than for in vivo therapy. In any case, a promising study in three newborn CF pigs has recently shown that 2 weeks after lentiviral delivery by aerosolization, the anion channel defect can be corrected in a large animal CF model [42]. Other recent studies assessed with pseudotyped lentivirus vectors in both murine lungs and human air-liquid interface cultures showed that preexisting and acquired immune responses do not interfere with vector efficacy [43]. In such study, at least 14% of the airway cells were transduced. Interestingly, toxicological results, notably the integration site profile showing absence of integration near oncogenic loci, sup-

Although the use of viral-based vectors in clinical trials still predominates over that of nonviral vectors, in recent years, there has been a notable increase in preclinical studies using nonviral vectors [44]. The reason is that these systems represent a safer, cheaper, and easier to produce alternative to viral-based vectors [18]. The main advantages of nonviral vectors include, among others, the ability to produce them on a large scale with high reproducibility and low cost; their relative stability after storage; the possibility of multiple-dose regimen administration due to their low immunogenicity; their high capacity to carry genetic material, independently of the size [45]; as well as the possibility to modify them chemically in order to regulate important physicochemical parameters, such as size, charge, morphology, or polydispersion, which clearly influence their final biological properties. All these important advantages have raised the interest of the scientific community to develop new biocompatible materials of different structures, compositions, sizes, and characteristics to transport therapeutic genes into specific organs or cells, overcoming the different extra- and

bacterial regions from AAV vectors [38, 39].

26 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

port further progression toward clinical trials.

**3.2. Nonviral vectors**

intracellular barriers [46].

In order to reach the nucleus of target cells and initiate transgene expression, the genetic material must overcome some extracellular and intracellular barriers, which will be discussed in this section, along with the most relevant strategies that have been developed to make the transfection process more efficient.

## **4.1. Extracellular barriers**

Even though intravenous injection is one of the most commonly used administration routes, especially for delivering genetic cargo into cancerous cells, some barriers still hamper its use in clinical practice, particularly in the CF disease [53]. First of all, the genetic material needs to be protected against extracellular enzymatic digestion, since DNA is quickly degraded when administered alone. To avoid such quick degradation, nonviral vectors based on both positively charged lipids and polymers offer the possibility to condense on their surface the genetic material by electrostatic interactions and minimize such undesirable effect [54]. However, the final positive charge of polyplexes or lipoplexes can interact in a nonspecific way, not only with target cell membranes but also with other negatively charged components such as serum proteins that jeopardize transfection efficiency [55]. These interactions could result in the formation of aggregates that classically are eliminated from the blood by the reticuloendothelial system. Interestingly, the incorporation of polyethylene glycol (PEG) motifs into the formulation of some nonviral carriers enhances the stability of complexes, since the highly hydrophobic nature of PEG chains creates a steric barrier to prevent both aggregation of complexes in blood circulation and extracellular enzymatic degradation by nucleases [55]. In any case, other relevant aspects related with both the length and the degree of PEGylation should be also considered, as they can also decrease DNA condensation efficiency with nanoparticles [56]. In addition to PEG, other polymers with hydrophobic nature such as poly(4-acryloylmorpholine) or poly(N,N-dimethylacrylamide) have recently emerged as interesting and promising alternatives to compensate or ameliorate the negative effects associated with PEGylation [57].

such biological compartment quickly before being degraded by the acidic environment and the enzymes found in lysosomes [63]. To avoid this enzymatic degradation, some interesting strategies can be used, such as the formation of pores in the endosome membrane by incorporation of amphiphilic cationic peptides. This creates strong internal tensions in the membrane and enhances the exit of endosome content through such pores [64]. Another strategy is known as the "proton sponge effect," where the low pH within the endosome allows the protonation of trapped compounds that have a large capacity to absorb protons from

Gene Therapy for Cystic Fibrosis: Hurdles to Overcome for Successful Clinical Translation

http://dx.doi.org/10.5772/intechopen.79719

and water in the endosome, which produce a swelling effect and its rupture. This effect has been observed in some cationic polymers with high buffering capacity over a wide pH range [64]. In CVME, internalized molecules go to the caveosome instead of lysosome [61] avoiding lysosomal degradation; however, there is still ongoing debate, with some authors claiming

Phagocytosis is a special type of endocytosis used mainly by macrophages, monocytes, neutrophils, and dendritic cells, although other cell types can also use this cellular entry pathway [61]. Endocytosis mediated by phagocytosis comprises the formation of membrane extensions with certain forms to capture particles generally greater than 1 μm. In contrast, for endocytosis mediated by macropinocytosis, membrane extensions do not surround particles but form some kind of protuberances that finally fuse with the cytoplasmic membrane. In many cases, the physicochemical properties of nonviral vector-based nanoparticles, such as particle size, superficial charge, morphology, or polydispersity, directly influence the endocytosis mecha-

Once the DNA is released into the cytosol of cells, it must enter the nucleus to produce its effect. This is considered a significant barrier that nonviral vectors must overcome in order to mediate a good transfection efficiency. One commonly used strategy to enhance nuclear import of genetic material is to incorporate a nuclear localization signal (NLS), such as polylysine or protamine [66]. NLS contains some amino acids that interact with some proteins of the cytoplasm known as importines. These importins enhance nuclear entry through the nuclear pore complex of the nuclear membrane through an energy-dependent mechanism [67].

The airways seem to be the natural way to treat respiratory diseases and a good alternative to systemic and more invasive procedures. Currently, aerosolization is the prefered method of administration for airway targeting since it is a noninvasive route that induces little stress to patients. Moreover, high quantities of drug can be deposited directly and fast into the lungs, which circumvents the blood circulation and avoids the first-pass effect of the liver. However, the effectiveness of such approach strongly depends on the development of smart drug formulation strategies. One of the critical steps that need to be taken into account for a successful gene delivery approach by inhalation is the formulation of the drug molecules into an appropriate inhalable form with sufficient stability and adequate aerodynamic properties [68]. Highly susceptible molecules, such as nucleic acid, require special attention when delivered by this route of administration. The physicochemical constraints

 and Cl− )

the medium (buffer effect). Such protonation causes an important entry of ions (H<sup>+</sup>

that CVME can fuse with lysosomes [65].

nism and consequently the transfection efficiency.

**5. Drug formulation**

Since intravenous injections present relevant hurdles that hamper the delivery of genetic material into target lung cells, local administration into the lung seems to be a reasonable alternative. In this case, the presence of mucus and the clearance mechanism are the most relevant barriers to overcome [58]. To avoid such barriers associated with pulmonary administration, other interesting noninvasive routes of administration, such as intranasal instillation, can be used to target lung cells. Nevertheless, the main problem is the low amount of genetic material that can be administered by the intranasal route. From a technical point of view, aerosolized nonviral vector/DNA complexes, carefully designed for inhalation in combination with appropriate excipients to enhance both particle flow and aerodynamic diameter, could be an interesting option since they are needle-free systems able to deliver locally high cargo concentrations [53].

To circumvent the diffusion of complexes into lung cells due to unspecific interactions with the biopolymer network of the mucus, some mucolytic agents that hydrolyze mucins can be added [58]. Other strategies include the incorporation of N-acetylcysteine to reduce disulfide bridges between the subunits of mucin, and consequently the viscosity [59], or the functionalization of nonviral vector formulations with mucolytic agents.

#### **4.2. Intracellular barriers**

Once extracellular barriers are overcome, there is still a long way full of hurdles before reaching the nucleus of target cells. Firstly, complexes carrying the genetic material need to be endocytosed by target cells. The interaction between complexes and cell membranes can occur in an unspecific way or can be mediated by a specific ligand, which is the preferred one, especially for in vivo applications [60]. Of note, the choice of ligand to be incorporated into the nanoparticle formulation depends not only on the target cell but also on the type of cell entry pathway that will be used once the ligand binds to the desired receptor.

Classically, there are four main pathways of endocytosis: clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CVME), phagocytosis, and macropinocytosis [61, 62]. CME is an energy-dependent mechanism widely studied and characterized [63]. Typically, this pathway is directly associated with lysosomes, where the genetic material needs to leave such biological compartment quickly before being degraded by the acidic environment and the enzymes found in lysosomes [63]. To avoid this enzymatic degradation, some interesting strategies can be used, such as the formation of pores in the endosome membrane by incorporation of amphiphilic cationic peptides. This creates strong internal tensions in the membrane and enhances the exit of endosome content through such pores [64]. Another strategy is known as the "proton sponge effect," where the low pH within the endosome allows the protonation of trapped compounds that have a large capacity to absorb protons from the medium (buffer effect). Such protonation causes an important entry of ions (H<sup>+</sup> and Cl− ) and water in the endosome, which produce a swelling effect and its rupture. This effect has been observed in some cationic polymers with high buffering capacity over a wide pH range [64]. In CVME, internalized molecules go to the caveosome instead of lysosome [61] avoiding lysosomal degradation; however, there is still ongoing debate, with some authors claiming that CVME can fuse with lysosomes [65].

Phagocytosis is a special type of endocytosis used mainly by macrophages, monocytes, neutrophils, and dendritic cells, although other cell types can also use this cellular entry pathway [61]. Endocytosis mediated by phagocytosis comprises the formation of membrane extensions with certain forms to capture particles generally greater than 1 μm. In contrast, for endocytosis mediated by macropinocytosis, membrane extensions do not surround particles but form some kind of protuberances that finally fuse with the cytoplasmic membrane. In many cases, the physicochemical properties of nonviral vector-based nanoparticles, such as particle size, superficial charge, morphology, or polydispersity, directly influence the endocytosis mechanism and consequently the transfection efficiency.

Once the DNA is released into the cytosol of cells, it must enter the nucleus to produce its effect. This is considered a significant barrier that nonviral vectors must overcome in order to mediate a good transfection efficiency. One commonly used strategy to enhance nuclear import of genetic material is to incorporate a nuclear localization signal (NLS), such as polylysine or protamine [66]. NLS contains some amino acids that interact with some proteins of the cytoplasm known as importines. These importins enhance nuclear entry through the nuclear pore complex of the nuclear membrane through an energy-dependent mechanism [67].
