**6. Commercialization**

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

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

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

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

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

porous hollow particles [71].

successful.

others [78].

delivery of nucleic acids by aerosolization.

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 available medical products based on gene therapy along with cell therapy and tissue engineering are classified as advanced therapy medicinal products. Although highly promising, their translation into clinical practice is nowadays hampered by major critical issues such as complex regulatory and ethical aspects, along with the intrinsic difficulties to scale up these products to an industrial level [20].

represents a great challenge that needs to be overcome for successful clinical application. In this sense, pilot plants, which employ small volumes of the product, represent an interesting option to gain knowledge on the technical process before full scale up production.

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

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

Despite the fact that the CFTR gene was cloned two decades ago, the current, conventional treatments for CF focus on masking the main symptoms, rather than addressing the underlying genetic cause of the disease. In this sense, gene therapy represents a promising alternative to tackle CF, considering the autosomal recessive nature of the most relevant ΔF508 mutation. Although the main objective of gene therapy seems simple, there are some hurdles that need to be overcome before gene therapy for CF becomes a realistic treatment option. In any case, the increase in knowledge and recent advances in biopharmaceutical technology offer reasonable hope for the treatment of this devastating disease. The minicircle technology, along with the new gene editing tools, offer important advantages compared with classical plasmids used to add functional copies of the gene. Additionally, intense research in novel nonviral vectors functionalized to overcome both extra- and intracellular barriers and the possibility to aerosolize such formulations without losing activity

This project was supported by the Basque Country Government (Department of Education, University and Research, predoctoral grant PRE\_2016\_2\_0302 and Consolidated Groups, IT907- 16) and by the University of Basque Country UPV/EHU (predoctoral grant PIF17/79). Authors wish to thank the intellectual and technical assistance from the ICTS "NANBIOSIS," more specifically by the Drug Formulation Unit (U10) of the CIBER in Bioengineering, Biomaterials,

and Nanomedicine (CIBER-BBN) at the University of the Basque Country (UPV/EHU).

Myriam Sainz-Ramos1,2†, Nuseibah AL Qtaish1,2†, Idoia Gallego1,2, Ilia Villate-Beitia1,2,

\*Address all correspondence to: joseluis.pedraz@ehu.eus and gustavo.puras@ehu.eus

1 NanoBioCel Group, University of the Basque Country (UPV/EHU), Spain

2 Biomedical Research Networking Centre in Bioengineering, Biomaterials and

Tania López1,2, Gustavo Puras1,2\* and José Luis Pedraz1,2\*

Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain

† These authors contributed equally to the work.

**7. Conclusion**

merit special attention.

**Acknowledgements**

**Author details**

Regarding the regulatory concerns of GTMP that affect clinical applications, the economical investments, along with their manufacture and control, demand more attention than chemically synthesized small molecules [86]. Therefore, a deep analysis of both costs and benefits needs to be done before considering the commercialization of such therapies [87].

Another relevant concern that jeopardizes the clinical use of GTMP in CF is the ethical aspect of clinical trials. Since the early 1990s, more than 25 Phase I gene therapy clinical trials have been conducted. These trials have been carried out largely to assess the safety and feasibility of gene transfer methods and their expression in the host, reporting variable successes for both viral and nonviral approaches. Gene therapy products designed for the treatment of CF must meet certain requirements in order to become a viable therapeutic option. For instance, their clinical efficacy must be demonstrated by analyzing appropriate variables of the lung function such as the patient´s vital capacity that they are able to expire in the first second of forced expiration (FEV1), their age, sex or body composition, and the therapeutic efficacy which must be maintained with repeated administrations. In addition, the GTMP must demonstrate an acceptable profile when it comes to side effects, and other considerations such as treatment of early versus established lung disease must also be analysed.

Since a high percentage of patients affected by CF are children, clinical trials involving these patients must carefully balance the potential benefits of these therapies and the associated risks [88]. Regarding this controversial issue, the Gene Therapy Advisory Committee recommends that clinical trials on children should only be performed under specific circumstances, whereby: (i) it has been demonstrated that the research is necessary to promote the health of the trial population, (ii) the research cannot be done in adults, and (iii) there is a high potential of therapeutic benefit [88]. In fact, owing to a demonstrated benefit of early gene therapy intervention, the age of enrolment of children in clinical trials has progressively reduced over the years from 18 to 12 years old. However, parents should have legal rights to make the final decision on behalf of their children.

Another critical hurdle that strongly compromises the clinical application of gene therapy products for the treatment of CF is the difficulty to scale up formulations that were originally developed for basic clinical research [89]. Most of these products are usually developed by small- and medium-sized enterprises, in collaboration with academic groups, which are usually highly engaged in preclinical activities, but have limited manufacturing experience at industrial level. For instance, the normal procedure for preparing nonviral-based gene therapy products is by simply mixing and pipetting the negatively charged genetic material and the positively charged polymer - or lipid-based nonviral vector formulations, which are often produced in the laboratory at small volumes that usually oscillate between 1 and 5 mL. However, the standardization of this procedure at industrial level to produce high and stable levels of complexes under GMP conditions represents a great challenge that needs to be overcome for successful clinical application. In this sense, pilot plants, which employ small volumes of the product, represent an interesting option to gain knowledge on the technical process before full scale up production.
