**10. Conclusion**

nanoparticles (MPP) may provide an opportunity to further overcome this barrier [65]. Drugloaded MPPs with non-adhesive coatings have been shown to penetrate mucus layers at rates nearly as fast as pure water. These developments may allow the penetration of delivery vehicles to airway epithelium, without reducing the protective function of the mucus itself. Furthermore, adjuvant regimens of N-acetylcysteine (NAC) with or without recombinant human DNase (rhDNase) were used to increase diffusivity of nanocomplexed, non-viral gene delivery vectors through sputum layers [66]. This strategy was able to increase gene expression by ~12-fold, making it another potential avenue for improving targeting in the lungs of CF patients. Complexes of pDNA or mRNA with GL67:DOPE:DMPE-PEG5000 (GL67) liposomes have also been described as a potential avenue for augmenting non-viral respiratory gene transfer [67]. Overall, developments in nanoparticle technology combined with advancements

Route of administration may be an important consideration as well, especially given the tendency for inhaled therapeutics to be entrapped in the mucus layer. Intratracheal highpressure spraying approaches have been effective in targeting airway epithelial cells in preclinical models [30,31,37], although efficiency is likely to decrease in the face of CF sputum. Preliminary evidence supports the claim that intravenous routes of administration may also target airway cells efficiently, while avoiding the barriers to a direct airway approach.

The continued development of humanized animal models of CF, including mouse, pig, and ferret models, will further our ability to investigate novel therapeutic strategies [68,69]. An early mouse model, *CFTRtm1UNC*, knocked out murine *CFTR* through a stop codon in exon 10; however, these mice showed a drastic drop in survival rates due to severe intestinal obstruction [70,71]. To overcome lethal intestinal defects, the mice were then 'gut-corrected' with a human *CFTR* construct driven by an intestinal-specific FABP promoter [72]. Studies in the FABPhCFTR/*Cftrtm1UNC* gut-corrected model have demonstrated that the human CFTR protein is indeed functional in mice. Using this or other models as a foundation, it may be possible to introduce a transgenic construct containing a mutated human *CFTR* driven by a lung-specific promoter. Creating humanized mice expressing the *CFTR*-∆F508 mutation, for instance, may offer an excellent tool for studying gene correction using nucleases and repair templates

In addition to the development of novel animal models, the identification of human lung stem cell populations has offered new hope for overcoming the issue of lung cell turnover [73]. If genome-editing vehicles could be efficiently targeted to lung stem cell populations, such as bronchioalveolar stem cells (BASCs) [74], Clara cells [75], or alveolar type II (ATII) progenitors [76], HDR in these self-renewing populations could support indefinite CFTR production.

Engineering strategies to minimize the risk of off-target cleavage and donor integration will also continue to be an important area of development. Along these lines, it will be critical to more thoroughly define standardized parameters for measuring off-target effects. State-of-theart techniques that can be used for measuring outcome parameters will also aid in assessing overall efficacy. Combining efforts to overcome these barriers to lung targeting, cell turnover, proper animal models, and off-target effects will enable the field to make continued progress

toward a novel gene correction strategy for the treatment of Cystic Fibrosis.

in aerosol-delivery devices may hold promise for the field.

372 Cystic Fibrosis in the Light of New Research

designed for direct translation to the clinic.

Over two decades since the cloning of the CFTR gene, numerous strategies have been inves‐ tigated to identify clinically relevant genetic variants, target cells of the airway, and overcome deleterious mutations. Rather than masking symptoms of the disease, novel therapies strive to address the underlying genetic cause of the Cystic Fibrosis phenotype. Agents have approached this goal with varying strategies, including attempts to overcome the patient's functional CFTR defects, supplement their cells with a functional copy of the protein, or directly repair genomic mutations at their source. Innovations in viral and non-viral delivery vehicles and methods for overcoming barriers to lung targeting have allowed for promising progress in recent years. Coupled with novel genome-editing reagents, such as ZFNs, TALENs, and the CRISPR/Cas9 system, the promise of a novel therapeutic approach is becoming an increasingly attainable goal within the field. Further advancement in minimizing off-target activity, increasing the efficiency of site-specific cleavage, and optimizing robust, transient, non-integrating nuclease delivery vehicles will bring us closer to achieving stable modification of the genome in the race toward *in vivo* gene correction of Cystic Fibrosis.
