**Acknowledgements**

incorporating positive feedback loops are estimated to increase strength of weak but highly

Besides increasing transfection efficiency through targeting and other strategies, careful consideration of toxicity, manufacturing and regulatory issues of non-viral delivery systems is mandatory. Scalability and long-term storage requirements are essential factors to be taken into serious consideration when developing non-viral formulations for potential commercial application and introduction in clinical practice [19]. A generally accepted advantage of nonviral vectors is their ease of large-scale manufacture. However, this can become more complex as formulations increase in complexity, incorporating stabilizing components and bioactive

Regarding toxicity of non-viral vectors, characteristics such as size, charge, surface function‐ alization, shape, and architecture may contribute to the toxicity profile of nanoparticles. Nonviral vectors are thought to cause toxicity through different mechanisms, including membrane destabilization and lysis, inducing oxidative stress, initiating inflammatory response, inducing global changes in gene expression profiles, among others [85]. Also, the properties of the biomaterials used can influence toxicity, depending on the rate of degradation and persistence in organs [19]. Persisting and accumulating biomaterials are more likely to induce an inflam‐ matory response; also, products of the degradation of nanoparticles could potentially cause toxicity. However, knowledge about how non-viral vectors are disassembled and metabol‐ ically processed is still very scarce and further research is necessary in this regard [19].

To conclude, the concept of a unique universal non-viral vector is nowadays abandoned, and it is increasingly accepted that future non-viral gene delivery platforms will be based on multifunctional vectors specifically tailored for different applications [1]. However, there are some generally assumed features that all non-viral vectors should accomplish for efficient gene delivery. In short, three main factors should be taken into consideration when developing nonviral gene delivery platforms: (i) formulation components (of both the vector and the DNA) – the nanoparticle should be able to protect DNA over extra- and intracellular barriers and deliver the cargo into the nucleus of target cells; once inside the nucleus, the plasmids can be addressed to the nuclear matrix for episomal replication and sustained expression; (ii) manufacturing issues – non-viral vector formulations should have potential for scale-up; and (iii) safety and regulatory issues – formulations should be non-toxic, non-immunogenic and should have suitable storage conditions. Although non-viral vectors are still far from clinical practice, they represent a safer alternative to conventional viral vectors. Several formulations and strategies are under investigation with the aim of overcoming extra- and intracellular barriers, enhancing targeted transfection and, in general, increasing transfection efficiency. In addition, those formulations would ideally be suitable for administration through noninvasive routes, such as the intranasal administration to target the brain, topical ocular administration for the retina and aerosols for pulmonary diseases. Finally, the inclusion of novel functional modules within both the carrier and the DNA molecule will produce a range of non-viral vectors tailored for specific applications, including the safe and long-term expression of therapeutic genes in humans [40]. Therefore, reasonable hope suggests that next-

specific regulatory elements [85].

170 Gene Therapy - Principles and Challenges

targeting ligands.

**4.3. Toxicity, manufacturing and regulatory issues**

This project was partially supported by the University of the Basque Country UPV/EHU (UFI 11/32), the Basque Government (Department of Education, University and Research, predoc‐ toral PRE\_2014\_1\_433 and BFI-2011-2226 grants), the National Council of Science and Tech‐ nology (CONACYT), Mexico, Reg. # 217101 and the Spanish Ministry of Education (Grant SAF2013-42347-R). Authors also 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 & Nanomedicine (CIBER-BBN) at the University of Basque Country (UPV/EHU).
