**3. Vectors**

bacterial region, known as pGM169 [8]. In such study, treated patients exhibited modest but significant improvements in lung function compared to placebo-treated ones during 1-year follow-up [19]. In any case, despite the optimism generated, there are still some concerns that need to be considered, such as the reproducibility of the results; the intensity of the response, probably conditioned by the degradation of formulation after aerosolization process; or the

Both previously mentioned approaches based on pDNA and mcDNA technologies allow to restore the function of the mutated CFTR gene, with the addition of normal copies, but they do not correct the mutation at their local chromosomal location. However, genome editing tools based on zinc-finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs), can specifically correct CFTR gene mutations at their natural chromosomal location, and so, the corrected gene can remain under the control of its endogenous promoter [20]. ZFNs are synthetic restriction enzymes, which have three or more zinc-finger DNA-binding motifs linked to the FokI restriction enzyme that recognizes trinucleotides in a specific DNA sequence [21]. When FokI enzyme creates a double-strand break (DSB) near the mutation place, cellular DNA repair mechanisms are activated to maintain cell viability. In these conditions, a donor DNA sequence with high 5′ and 3′ homology with the DNA sequence where DSB has been generated can be exogenously supplemented to enhance the correction of the mutation by homologous recombination (HR) mechanism. This genome editing tool has been successfully used in vitro to correct CFTRΔF508 mutation in both human bronchial epithelial

TALEN technology is very similar to ZFNs. These nucleases were originally characterized in *Xanthomonas* bacteria, in which TALEN proteins are secreted when *Xanthomonas* infect a wide variety of plants, thus activating genes that help to develop the pathogenesis. This genome editing tool also produces a DSB around the mutation site of the target gene and consequently induces cellular DNA repair mechanisms [24]. TALENs are considered as a more efficient and cost-effective alternative to ZFNs [25]. In the case of ZFNs, each finger module recognizes three to four bases of the DNA sequence. However, in the case of TALENs, gene recognition is mediated by a more specific mechanism, where each module of 33–35 amino acid targets a single nucleotide. This technology has been recently applied to correct CFTRΔF508 mutations in CF patient-specific IPS cells [26]. Overall, such study reported correction of patient-specific IPS cells in less than 3 months, which could allow rapid scaling up for future applications.

Clustered regularly interspaced short palindromic repeats (CRISPR) methodology, originally described as an adaptive immune response in archaea, follows the same rationale described for ZFNs and TALENs, but instead of protein domains, short RNA molecules are used to drive the required homology [27]. In this case, an endonuclease called Cas9 is guided by a single guide RNA (gRNA) to hybridize specifically with the mutated sequence in the DNA; then, as described for ZFNs and TALENs, the resulting DSB triggers cellular DNA repair mechanism [28]. The main advantage of CRISPR technology is that it is an easy-to-synthesize costeffective tool that is able to correct more than one mutation at the same time, if multiple-gene

number of patients that received such treatment.

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

cells [22] and CF-induced pluripotent stem (IPS) cells [23].

**2.3. Genome editing tools**

One of the main concerns related to the clinical application of gene therapy is the design and development of safe and effective gene delivery vectors to introduce exogenous genetic material into the nucleus of target cells [30, 31]. In the absence of gene delivery vectors, naked genetic material is quickly degraded mainly by exogenous deoxyribonuclease enzymes, which clearly inhibit transfection efficiency [31]. Additionally, the negatively charged genetic material, mainly due to the phosphate groups, hampers the electrostatic interactions with cell membranes, which are negatively charged too. Therefore, the clinical application of gene therapy demands the design, characterization, and evaluation of efficient and safe carriers to mammalian cells.

#### **3.1. Viral vectors**

At present, viral-based carriers are the most appropriate from an effectiveness point of view. The natural evolution that viruses have undergone over many years has allowed them to face different intra- and extracellular barriers and, consequently, infect target cells with high efficiency.

In the CF field, a wide variety of viral-based vectors has been developed in clinical trials. The first one was performed in 1993 with adenovirus in three patients, where partial correction of the chloride transport in nasal epithelium was observed [32]. Some of the main advantages of adenoviruses include their non integrating nature and their natural tropism for the lung. However, despite such favorable properties, and the high transduction efficiency observed in most tissues, gene expression usually remains transient, and these viruses can induce strong immune and inflammatory responses in a dose-dependent manner, which clearly brings up safety issues and, therefore, limits their application in the clinical practice [8].

Initial clinical trials performed with adenovirus allowed the development of adeno-associated viruses (AAV), which have interesting characteristics for their application in gene therapy, such as broad tissue tropism, high transduction efficieny, and persistent episomal expression, which can last for years, even though it is a non integrating vector [33, 34]. In addition, recombinant AAV vectors have been shown to be safe in several clinical trials, as they are not related to any known human disease. However, these vectors also present relevant limitations, the main one being their low capacity to load genetic material (<5 kb) [35]. Between 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 bacterial regions from AAV vectors [38, 39].

Within the large variety of nonviral vectors developed, most of them are based on peptides as well as on cationic lipids and polymers, which form the corresponding complexes (polyplexes and lipoplexes) after electrostatic binding with DNA [47]. The resulting complexes protect nucleic acids from enzymatic degradation and facilitate cellular uptake by interactions with the cytoplasmic membrane [48]. The PEG-CK30 peptide, due to its low immunogenicity and its ability to be endocytosed by cells, is one of the most widely used, although the formulation

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

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

must be optimized to allow its administration in aerosol form to reach the lungs [49].

which PEI/DNA concentrates are prepared.

**4. Biological barriers**

**4.1. Extracellular barriers**

transfection process more efficient.

Regarding cationic polymers, polyethylenimine (PEI) is one of the most used, since its chemical structure can be easily modified to increase the efficiency of transfection, for example, by incorporating lactose (Lac-PEI) to improve intracellular trafficking [50]. However, PEI has not yet been used in any clinical trial. The main limitation lies in the difficulty that exists to prepare PEI polyplexes at high DNA concentrations [49]. One of the most promising strategies that have been used to circumvent this problem is the use of ultrafiltration methods, through

In the case of cationic lipids, some of the most widely used to develop nonviral formulations in the CF field are N-[1-(2,3-dioleyloxy) propyl]-N, N, N-trimethylammonium, dioleoylphosphatidyl ethanolamine, and dioleoyl trimethyl ammonium [51]. However, currently, the most promising nonviral vector in CF clinical trials is based on the cationic lipid GL6TA, which was synthesized to prevent DNA degradation in the lysosome and to be stable after pulmonary administration by aerosolization [8, 51]. In 2011, a preclinical comparative study of the use of PEI, PEG-CK30, or GL67A nonviral vectors in aerosols demonstrated that this last formulation was the best one to transport DNA to sheep's lungs [7]. Moreover, in an extensive preclinical study performed in 2014 [52], it was corroborated that the formulation based on the lipid GL67A, which had already been used for 15 years in CF clinical trials, was still suitable

for administration in multiple-dose regimen, without any observed loss of activity [8].

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

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].

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, support further progression toward clinical trials.

#### **3.2. Nonviral vectors**

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 intracellular barriers [46].

Within the large variety of nonviral vectors developed, most of them are based on peptides as well as on cationic lipids and polymers, which form the corresponding complexes (polyplexes and lipoplexes) after electrostatic binding with DNA [47]. The resulting complexes protect nucleic acids from enzymatic degradation and facilitate cellular uptake by interactions with the cytoplasmic membrane [48]. The PEG-CK30 peptide, due to its low immunogenicity and its ability to be endocytosed by cells, is one of the most widely used, although the formulation must be optimized to allow its administration in aerosol form to reach the lungs [49].

Regarding cationic polymers, polyethylenimine (PEI) is one of the most used, since its chemical structure can be easily modified to increase the efficiency of transfection, for example, by incorporating lactose (Lac-PEI) to improve intracellular trafficking [50]. However, PEI has not yet been used in any clinical trial. The main limitation lies in the difficulty that exists to prepare PEI polyplexes at high DNA concentrations [49]. One of the most promising strategies that have been used to circumvent this problem is the use of ultrafiltration methods, through which PEI/DNA concentrates are prepared.

In the case of cationic lipids, some of the most widely used to develop nonviral formulations in the CF field are N-[1-(2,3-dioleyloxy) propyl]-N, N, N-trimethylammonium, dioleoylphosphatidyl ethanolamine, and dioleoyl trimethyl ammonium [51]. However, currently, the most promising nonviral vector in CF clinical trials is based on the cationic lipid GL6TA, which was synthesized to prevent DNA degradation in the lysosome and to be stable after pulmonary administration by aerosolization [8, 51]. In 2011, a preclinical comparative study of the use of PEI, PEG-CK30, or GL67A nonviral vectors in aerosols demonstrated that this last formulation was the best one to transport DNA to sheep's lungs [7]. Moreover, in an extensive preclinical study performed in 2014 [52], it was corroborated that the formulation based on the lipid GL67A, which had already been used for 15 years in CF clinical trials, was still suitable for administration in multiple-dose regimen, without any observed loss of activity [8].
