**2. Genetic material**

#### **2.1. Plasmid DNA**

According to recent reports, in the European Union, 1 of every 2000–3000 newborns is affected by CF. In the USA, the incidence is 1 per 3500 births. In Asia, the predisposition to CF is low; however, there is evidence to show that this disease is severely underdiagnosed [2]. The basic characteristic of CF is the transport of defective ions in the apical membrane of most secretory cells, which leads to an altered secretion of mucus in the epithelium of the respiratory tract, the digestive tract, the pancreas, the liver, and the reproductive track [1]. The conventional treatments available on market, which include, among others, antibiotics, pancreatic enzyme supplements, high-fat diets, and even physiotherapy [3], afford the consequences derived from CFTR dysfunction and have significantly improved the mean life expectancy of patients affected by the disease up to 34 years [4]. However, their quality of life is severely compromised mainly due to side effects and interactions among such treatments [5]. Therefore, other therapeutic options such as gene therapy, in which the main goal is to restore the function of the mutated CFTR protein acting on the genetic cause of the problem, need to be considered. CFTR gene was cloned more than two decades ago, and the monogenic and autosomal recessive nature of CF disease means that the addition and expression of the corrected gene could reverse the underlying cause of the disease. Therefore, there is reasonable hope to consider gene therapy as a potential realistic medical option, and consequently, some clinical trials have been performed since 1993. However, despite the moderate optimism that emerged with the development of such clinical assays, there are still some hurdles to overcome before considering gene therapy a realistic medical option. Main concerns are related to the intrinsic properties of genetic materials, the development of safe and efficient gene delivery vectors able to deliver genetic materials to the nucleus of target cells, the design of a drug formulation suitable for pulmonary gene delivery applications, and the hurdles associated with the

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

**Figure 1.** Hurdles that gene therapy should overcome in order to reach clinical practice in the treatment of CF disease.

commercialization of such drugs (**Figure 1**).

Bacterial plasmid DNA (pDNA) remains an interesting biomolecule for gene transfer, with several promising reports and clinical trials in progress worldwide [6]. In CF, pDNA has been successfully delivered by nonviral vectors to the sheep lung [7]. Additionally, when administered in multiple-dosage regimen, no loss of activity was observed [8]. In order to be produced in recombinant bacteria and to express their therapeutic gene of interest (GOI), pDNA needs a bacterial origin of replication sequence (bac-ORI). In addition, pDNA backbone includes a sequence with resistance to one/various antibiotics such as kanamycin, which allows to select the clone of bacteria transformed that expresses the plasmid. Finally, a eukaryotic promoter is needed to enhance GOI expression [9]. Usually, when pDNA reaches the nucleus of target cells, it remains in an episomal position, which means that it replicates independently from the host chromosomal DNA, avoiding the undesirable activation of oncogenic genes [10]. The main concerns of pDNA in gene therapy are related to safety issues. Classically, in eukaryotic cells, pDNA has been associated with the induction of undesired immune responses and secretion of proinflammatory cytokines [11]. For instance, a transient neutrophilic infiltration and an elevation in proinflammatory cytokines have been reported in mouse lung [12]. Although the episomal nature of pDNA could be an interesting advantage, the transfection efficiency remains compromised mainly by the transient and relatively low gene expression. Additionally, the size of the plasmid, determined by the number of base pairs, jeopardizes transfection efficiency [13, 14].

#### **2.2. Minicircle DNA**

In order to overcome the previously mentioned disadvantages associated with the use of pDNA in gene therapy, small plasmidic cassettes known as minicircle DNAs (mcDNAs) have been recently developed [15]. Cameron and Scheleff first employed mcDNA terminology in 1995. Nowadays, this technology offers a potential alternative to enhance both transfection efficiency and safety of gene delivery [14]. Basically, mcDNAs are circular constructors similar to pDNA but significantly smaller, since mcDNAs contain a minimal expression cassette, of a promoter, a transgene, and a polyadenylation, signal but are devoid of bacterial pDNA elements. Thus, mcDNA technology allows sustained transgene expression mainly due to a lower activation of nuclear transgene silencing mechanisms and reduced immunogenic responses in vivo [16, 17].

In the lung, some promising results have been obtained with the use of small plasmidic cassettes [18]. In fact, results of a Phase IIb double-blind clinical trial for CF have been recently reported. These trials were performed with a plasmid encoding CFTR gene and lacking CpG 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 number of patients that received such treatment.

targeted sgRNAs are delivered to target cell along with the Cas9 protein, which makes it an excellent option [24]. CRISPR technology has been applied to repair CFTRΔF508 mutations in

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

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

This study represents an interesting proof of concept for CFTRΔF508 correction by HR using CRISPR/Cas9 technology in primary adult stem cells derived from patients with a singlegene hereditary defect and offers reasonable hope to be successfully applied to the lungs of patients affected by CF. However, some relevant concerns, mainly related to the frequency of

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

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

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

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

safety issues and, therefore, limits their application in the clinical practice [8].

undesirable off targets, still need to be resolved in order to reach clinical practice [19].

intestinal stem cell organoids of CF patients [29].

**3. Vectors**

mammalian cells.

**3.1. Viral vectors**

efficiency.

#### **2.3. Genome editing tools**

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

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 targeted sgRNAs are delivered to target cell along with the Cas9 protein, which makes it an excellent option [24]. CRISPR technology has been applied to repair CFTRΔF508 mutations in intestinal stem cell organoids of CF patients [29].

This study represents an interesting proof of concept for CFTRΔF508 correction by HR using CRISPR/Cas9 technology in primary adult stem cells derived from patients with a singlegene hereditary defect and offers reasonable hope to be successfully applied to the lungs of patients affected by CF. However, some relevant concerns, mainly related to the frequency of undesirable off targets, still need to be resolved in order to reach clinical practice [19].
