**8. Hurdles to success: Limitations to gene correction for cystic fibrosis**

The lung has evolved with natural defense mechanisms against foreign pathogens. As such, a number of intracellular and extracellular barriers must be overcome in order to target new technologies to the lung [61]. In addition to this, the lungs of CF patients are even more difficult to target, owing to the increased airway mucus (sputum) lining the lungs. Especially in more progressed CF patients, thickened mucus linings have proven prohibitive in several gene replacement therapy approaches. Hida and colleagues reported that expectorated sputum from CF patients effectively traps and slows the diffusion of both Ad and AAV viral vectors [62]. For instance, where the sputum penetration of muco-inert nanoparticles is reduced by only 40-fold compared with pure water, that of Ad and AAV particles is slowed by 3,000-fold and 12,000-fold, respectively. Poor penetration of mucus layers may be a major component preventing effective viral gene replacement therapy or the use of viral vectors to deliver genome-editing components for CF.

nucleases would also minimize the long-term threat of off-target events associated with the

In a recent study, nuclease-encoding chemically modified mRNA (nec-mRNA) was described as a novel vehicle for delivering genome-editing components directly to the lung [37]. Using a murine model of SP-B deficiency, nec-mRNA-encoded ZFNs were able to demonstrate the

In the transgenic mouse model of SP-B deficiency, SP-B cDNA is under the control of a Tetracycline-inducible promoter [58]. Administration of doxycycline allows SP-B to be expressed at wild-type levels. If doxycycline is removed, SP-B expression drops and mice begin to exhibit phenotypic changes similar to those seen in humans with the disease: thickened alveolar walls, heavy cellular infiltration, increased macrophages and neutrophils, interstitial edema, congestion, augmented cytokines in the lavage, a significant drop in lung function,

In order to demonstrate the value of nec-mRNA for lung-based genome-editing applications, this report utilized HDR to insert a constitutive CAG promoter immediately upstream of the SP-B cDNA. The resulting doxycycline-independent expression was able to significantly

While this study was not performed in a humanized mouse model, the approach was able to demonstrate that extra-chromosomal nec-mRNA is capable of transducing airway epithelial cells, expressing genome-editing reagents, and achieving HDR rates sufficient for therapeutic

**i.** mRNA modification schemes can be customized to optimize expression and mini‐

**iii.** Complexing nec-mRNA to chitosan-coated nanoparticles can increase transduction

**v.** nec-mRNA-mediated ZFN delivery can facilitate HDR rates comparable to AAV-

**8. Hurdles to success: Limitations to gene correction for cystic fibrosis**

The lung has evolved with natural defense mechanisms against foreign pathogens. As such, a number of intracellular and extracellular barriers must be overcome in order to target new

**ii.** Intratracheal delivery of nec-mRNA is able to target airway epithelial cells.

**iv.** nec-mRNA expression is transient in comparison to AAV-encoded ZFNs.

first report of life-prolonging gene correction specifically within lung tissue.

and acute onset of respiratory distress leading to death within days [59,60].

levels of protein expression. Main findings from the study include:

use of stably expressing, and possibly integrating viral vectors.

**7. Gene correction in the lung**

370 Cystic Fibrosis in the Light of New Research

prolong the life of treated mice [37].

mize immunity.

encoded ZFNs.

efficiency in the lung.

Limits to stable expression of functional CFTR also play an important role. For gene replace‐ ment therapy approaches, this may include immune responses against the vector capsid, the inability to re-administer, as well as turnover of CFTR-expressing lung cells. For gene correc‐ tion approaches, transient expression of nucleases does not destabilize the downstream effects of HDR. However, lung cell turnover continues to be an issue, making the possibility of readministration important.

The levels of CFTR expression required to halt the progression of CF lung disease remain largely unknown, as well as the cell types most suited as targets. While airway epithelial cells are generally considered to be the ideal target, airway histology and entry receptor expression patterns may impact the ability of this cell type to be transduced. The absence of adenoviral entry receptors on the apical surface of airway epithelium, for instance, is one of the major reasons that adenoviral gene therapy vectors are no longer pursued. Furthermore, turnover of these terminally differentiated cells will eventually require therapeutics to be redelivered to new target cells.

In addition to the most relevant cell type, another question remains: is it preferable to obtain low levels of CFTR in a high percentage of cells, or high levels of CFTR in only ~10% of cells? Gene replacement and correction approaches are more likely to attain the latter, although it remains unknown whether this will be sufficient to show therapeutic effect. One recent study has shown that restoration of normal mucus transport rates in cultured CF human airway epithelial cells required at least 25% of surface epithelial cells to be targeted by CFTR gene replacement therapy [63]. Whether this figure will translate to clinical benefit in an *in vivo* setting remains to be seen.
