**5. Drug formulation**

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

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

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

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 functional-

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

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

ization of nonviral vector formulations with mucolytic agents.

that will be used once the ligand binds to the desired receptor.

associated with PEGylation [57].

cargo concentrations [53].

**4.2. Intracellular barriers**

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 such as the hydrodynamic shear forces generated during aerosolization can induce degradation of the nucleic acids, which will be more or less important depending on their size [69, 70]. Therefore, the need to develop a suitable formulation able to protect the material from degradation and at the same time ensure delivery of nucleic acid to the target cells in the lung needs to be deeply considered. In this sense, the commonly accepted aerodynamic size for pulmonary gene delivery is within the range of 1–5 μm. Larger particles (4–7 μm) tend to deposit in the airways, while smaller particles (1–3 μm) and those in submicron range (<1 μm) reach the lower airways and deeper lung [68]. The aerodynamic diameter of a particle can be modified not only by changing its size but also by varying its density or shape, which opens new possible strategies for gene delivery to the lung, such as the design of large porous hollow particles [71].

Suitable formulations for pulmonary delivery are mainly prepared either by dissolving or by suspending the therapeutic molecules in a liquid or formulating them into a dry powder for inhalation using liquid inhalers (including nebulizers), dry powder inhalers (DPIs), or pressurized metered dose inhalers (pMDIs); each of them is suitable for different applications. Once the aerosolized droplets or microparticles are deposited next to the target location into the lungs, they need to dissolve in the lung lining fluid for subsequent absorption and cellular uptake [72]. Nowadays, viral gene delivery to the lungs is limited to liquid formulations using a nebulizer [36], and there is no dry powder or metered dose inhaler formulation available for any vector-drug combination. In most cases, the gene transfer efficiency to lung cells using viral vectors is still too low with traditional nebulizer devices, probably due to the degradation of viral envelope by the shear forces caused during aerosolization [73] and the viscous mucus found in obstructive diseases, like CF [74]. Moreover, and as previously highlighted, the use of adenoviral or AAV vectors would likely induce an acute immune response upon the initial administration or result in low efficacy following repeat dosing. This is particularly relevant since aerosolized gene therapy might require repeat dosing because mucus clearance mechanisms and/or phagocytes may engulf and destroy the drug vector before it can be taken up by target cells [75]. In contrast, the simpler composition of nonviral vectors may have, in this case, an advantage over viral vectors, making readministration potentially more successful.

Although pulmonary gene-based therapies have not yet been granted marketing approval, numerous strategies are being tested both in vitro and in vivo, and various clinical trials are underway [19, 36]. **Table 1** summarizes some of the strategies used to date for the pulmonary delivery of nucleic acids by aerosolization.

The elaboration of DPIs, composed of drug-based dry powders and an aerosol-generating device, also presents important advantages such as high physicochemical stability, easy handling, and propellant-free aerosols. In order to transform the therapeutic nucleic acids into stable dry powders, several techniques, such as freeze-drying (FD) [79], spray-drying (SD) [80], and spray freeze-drying (SFD) [81], are being investigated. In addition, the incorporation of suitable stabilizing agents/thermal protectors such as polysaccharides (sucrose [79], trehalose [79], agarose [82], lactose [83], mannitol [81], or chitosan [84]), amino acids (leucine

**Description Vector Aerosolization References**

PEI Nebulization [68]

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

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

AAV2 viral Nebulization [32]

PEI Nebulization or instillation [70]

Chitosan Dry powder, obtained by SFD, administered by intratracheal syringe

PEI Dry powder in insufflator lyophilization/

Nebulization [69]

Multiple nebulizers [71]

Nebulization [14]

powderization with lactose, sucrose, or trehalose

[72]

[73]

In addition to the above concerns, other relevant issues specifically related to the commercialization of gene therapy medicinal products (GTMP) must also be considered. Commercially

[84] or glycine [82]), or proteins (BSA [85]) is critical.

**Table 1.** Pulmonary gene delivery strategies by aerosolization.

**6. Commercialization**

In vivo aerosol delivery of

Randomized, double-blind, placebo-controlled, Phase II trial in CF patients with mild

Nebulization of receptortargeted nanocomplexes for in vivo gene delivery to the airway epithelium

In vivo repeated aerosol delivery of pDNA/PEI complexes with CpG-free

In vivo aerosol delivery of DNA/liposomes to the lung

Randomized, double-blind, placebo-controlled, Phase

In vivo intratracheal administration of pDNAchitosan dry powders, obtained by SFD

Dry powder aerosols for in vivo gene delivery to the Receptortargeting peptides and cationic liposomes

GL67A cationic liposomes

GL67A cationic liposomes

PEI-DNA comp

lung disease

plasmids

IIb trial

lung

Nowadays, the most studied approach for gene delivery to the lung involves the nebulization of the selected formulation [76], turning it from a liquid solution to microdroplets. Depending on the aerosolization system used, such as jet, ultrasonic, or mesh nebulizers, the implemented hydrodynamic stress that the therapeutic molecules would be subject to varies [77]. Interestingly, several strategies have been studied to reduce the damage to the genetic material during the aerosolization process, by condensing the nucleic acids with positively charged molecules, such as polyethylenimine (PEI), protamine, or poly-L-lysine (PLL), among others [78].


**Table 1.** Pulmonary gene delivery strategies by aerosolization.

The elaboration of DPIs, composed of drug-based dry powders and an aerosol-generating device, also presents important advantages such as high physicochemical stability, easy handling, and propellant-free aerosols. In order to transform the therapeutic nucleic acids into stable dry powders, several techniques, such as freeze-drying (FD) [79], spray-drying (SD) [80], and spray freeze-drying (SFD) [81], are being investigated. In addition, the incorporation of suitable stabilizing agents/thermal protectors such as polysaccharides (sucrose [79], trehalose [79], agarose [82], lactose [83], mannitol [81], or chitosan [84]), amino acids (leucine [84] or glycine [82]), or proteins (BSA [85]) is critical.
