Successful gene transfer to CF airways in vivo has been principally hampered by a lack of efficacy due to the inefficiency of gene transfer to human airway epithelium that normally exhibits CFTR function 
. Although there is clear evidence that both airway surface and submucosal gland epithelia are dysfunctional in CF, presently, the precise airway regions of the CF lung that require CFTR delivery for restoration of normal physiological function and reduction of disease symptoms are not well established. Although dependent on antibodies used, CFTR has been localized to human ciliated cells 
and the fluid-secreting cells of the submucosal glands 
. Previously noted physiologic characteristics of ciliated cells also indicate that ciliated cells function to maintain airway surface hydration 
. Certainly, ciliated cells facilitate effective MCT and airway mucus clearance. Together, these properties of ciliated cells combined with the abundance of ciliated cells throughout the human airways make this cell type a logical, although not exclusive, target for CF lung gene delivery strategies.
In this study, we have shown that PIV-mediated CFTR delivery to ciliated cells is efficient and sufficient for correcting the CF airway epithelium phenotype, i.e., efficient delivery of CFTR to CF human ciliated airway epithelium corrected hallmark characteristics of CF HAE that mimic the initiating events of CF lung disease, i.e., abnormal ASL volume homeostasis and mucostasis ( and ). Abnormal ASL homeostasis in CF airway epithelium is due to dysregulated Na+
ion transport 
, both consequent to the absence/dysfunction of CFTR at the apical membrane 
. Here, we have demonstrated that delivery of CFTR to ciliated cells restores Cl−
secretion and reduces the Na+
hyperabsorption characteristic of the CF airway epithelium in vitro and in vivo, providing confirmatory evidence that CFTR functions as both a Cl−
channel and regulator of ENaC within ciliated cells. Further, we have demonstrated that correction of the ion channel defects of CF HAE restores the integrated physiology required for ASL regulation, which ultimately restores MCT (shown schematically in ).
A critical variable for restoration of CFTR functional activity by PIV is the percentage of cells expressing CFTR. Using CF HAE, we demonstrate that restoration of normal ASL height and MCT required CFTR delivery to approximately 25% and approximately 40% of surface epithelial cells, respectively. We suggest that restoration of ASL height is the most predictive measure for these studies, as PIV clearly exerts cytotoxic effects on MCT, but not ASL measurements. These effects were isolated to MCT, but not ASL, suggesting that virus-mediated cytotoxicity may affect the synchrony of cilia beat, leading to modestly reduced effectiveness of ciliated cells to transport mucus.
Previous studies with gap junction–coupled polarized, but not differentiated, airway epithelial cell lines suggested that approximately 6%–10% of cells required CFTR to correct the Cl−
transport defect 
, whereas almost all cells (>90%) required CFTR overexpression to correct ENaC hyperabsorption 
. Clearly, expression of CFTR in nonciliated airway epithelial cells would be predicted to increase fluid secretion onto the apical surfaces of these cells although these previously published studies did not test this hypothesis. In our studies, we have directly shown that expression of CFTR in 60% of CF ciliated cells fully corrects the ENaC hyperabsorption defect () and that CFTR expression in approximately 25% of cells (approximately 30% of ciliated cells) corrects ASL volume homeostasis in CF HAE (). With respect to ENaC activity after CFTR delivery, the reasons why our data differ from these previous studies are unclear, but it may be speculated that differentiated human airway epithelium models as used in this current study are more representative and relevant to the required efficiency of CFTR delivery to human airway epithelium in vivo.
Although our data indicate that CFTR delivery to CF ciliated cells is sufficient for restoring MCT to CF HAE, it is likely that delivery of CFTR to other nonciliated surface epithelial cells may provide functional CFTR activity capable of hydrating the airway surface. At present, we are not aware of gene delivery vectors capable of delivering CFTR exclusively to nonciliated cells of HAE to determine whether CFTR expression in nonciliated cells also restores MCT. In our studies, we have combined the requirement of ciliated cells for generation of MCT with CFTR targeting of CF ciliated cells to restore defective MCT. Since ciliated cells are the predominant airway surface epithelial cell type throughout the human conducting airways, vectors targeting at least ciliated cells may achieve the required efficiency of delivery for restoration of MCT. We propose that targeting at least ciliated cells provides efficient and effective CFTR function that is sufficient for restoration of MCT.
Our data using an in vitro model of human airway epithelium predict that CFTR delivery to 25% of CF airway epithelial cells will restore MCT to near normal levels. However, it remains to be determined what proportion of normal MCT rates in vivo would be beneficial to CF patients. Tracheal mucus velocities in young smokers are significantly reduced compared to young nonsmokers (3.4 mm/min versus 10.0 mm/min) but without significant differences in lung function, perhaps suggesting that MCT at rates below “normal” may be sufficient to maintain pulmonary healthy 
. These in vivo studies measured mucus velocities only in the trachea, whereas CF lung disease likely initiates in the more vulnerable, smaller bronchiolar airway regions. If these regions respond similarly to CFTR delivery, then it is possible that delivering CFTR to fewer than 25% of CF cells may provide sufficient MCT to maintain healthy airways. Further testing of this hypothesis will require appropriate in vivo studies.
Since PIV infects ciliated airway epithelium of hamsters and human and nonhuman primates, but not those of the murine airways, testing our PIV vectors in vivo in appropriate models is difficult. Additionally, it has been recently reported that expression of human or murine CFTR in ciliated cells of CFTR−/−
mice failed to correct the nasal epithelium bioelectric defect, although correction was demonstrated in neonatal, but not adult, tracheal epithelium 
. One explanation for these results, in contrast to our data, is that murine CFTR expressed in murine ciliated cells may not function correctly. Our study highlights the need to test vectors for CFTR delivery in appropriate human models and that such data obtained from CF mouse models require cautious interpretation. The recent generation of a CF pig model 
may be beneficial for testing such vector systems, but preliminary data using ciliated airway epithelial cultures derived from porcine trachea suggest that this species is also not infected by the human viruses from which our PIV vectors are generated.
Using our prototypic PIV vector, we could not determine the lowest limit of CFTR expression on a per cell basis required for correction since this vector significantly overexpressed CFTR relative to endogenous levels. In this regard, because CFTR is critical for regulation of ASL homeostasis, there has been concern that overexpression of CFTR in CF airways would “supercorrect” Cl−
transport and generate excessive fluid secretion. This concern, while reasonable, appears unwarranted based on our observations that ion transport rates and ASL heights in CF HAE after CFTR delivery did not exceed that measured in non-CF HAE (, , and ). These data agree with previously published reports in which CFTR was transgenically overexpressed in mouse airway epithelium without deleterious results in terms of cell or organ toxicity 
. However, in this transgenic study, CFTR was overexpressed in Clara cells and alveolar type II cells of the mouse lung, and so our study represents the first demonstration, to our knowledge, of the functional safety of CFTR overexpression in human ciliated cells. Since CFTR overexpression did not supercorrect ASL regulation, we speculate that normal airway epithelium exhibits multiple apical membrane regulatory mechanisms in addition to CFTR levels that prevent excessive secretion of fluid into the airway lumen, i.e., airway flooding.
Demonstrating that PIV expresses CFTR at levels in excess of those required to restore full function to CF HAE suggests that further attenuation of PIV will be feasible while still providing sufficient CFTR for functional correction. Indeed, the lower replication capacity 10-fold reduction of PIVCFTR compared to PIVGFP, in the context of >100-fold overexpression of CFTR in individual ciliated cells, suggests that further attenuation of PIV will continue to provide sufficient CFTR for correction of the CF MCT defect and possibly further reduce the generation of inflammatory mediators and cytotoxicity associated with our PIV vector prototype. The continued effort to develop vaccines against PIV has generated a wealth of live attenuated recombinant PIV 
that exhibit attenuated replication. It is interesting to note that PIV3 vaccine candidates have been extensively evaluated after lumenal airway delivery in adults and infants as young as 3 mo 
; an age of CF patients in which CFTR replacement would be desirable.
The demonstration of efficacious CFTR
gene delivery to human ciliated airway epithelium overcomes a major hurdle to gene transfer approaches for CF lung disease. Other strategies to improve gene delivery to the human airways are ongoing and are focused on vector development 
and/or manipulation of the host tissue 
. However, the results so far published have not shown significant improvement in the ability to deliver transgenes to human ciliated airway epithelium. Lentiviral-based vectors pseudotyped with Ebola, influenza virus, baculovirus, Sendai, or SARS-CoV envelope proteins efficiently transduce airway epithelial cells in vitro and murine airways in vivo 
, suggesting that combining useful envelope glycoproteins with the potential longer duration of gene expression afforded by lentiviruses may provide novel vectors for lung gene transfer strategies. Similar vectors can be envisioned using the glycoproteins of PIV to target lentiviruses to human ciliated airways. However, to date, none of these vector systems have progressed to functional studies after delivery of CFTR to human ciliated airway epithelium, and no demonstration of correction of the CF phenotype (e.g., ASL height or MCT) has been reported.
Collectively, the studies reported here demonstrate the efficiency of CFTR delivery to human CF ciliated airway epithelium that is sufficient to reverse the CF phenotype of ASL dehydration and mucostasis. Our prototypic PIV vector provides a useful tool for manipulating ciliated cell function and for investigating the future potential of delivering functional CFTR to the airways of CF patients.