**4.3. CF pig**

successfully deliver the transposon transgene [80]. More recently, the generation of a hybrid *piggyBac*/Ad and *piggyBac*/AAV facilitated delivery of a nonviral transposon. However, unlike SB, neither *piggyBac*/Ad nor *piggyBac*/AAV required an extra recombinase step for transposi‐ tion to occur [81]. In contrast to the hybrid SB*, piggyBac* has not exhibited overexpression inhibition or limitations on size of the genetic cargo [85]. *PiggyBac*/Ad hybrid vector success‐ fully delivered a CFTR expression cassette to primary airway epithelial cultures *in vitro* that corrected the anion transport defect up to 4 months in culture. In reporter gene studies,

transgene expression persisted for the 1-year duration of the experiment in mice [81].

Animal models serve an important testing ground for somatic cell gene transfer applications. Mice with null mutations [86-89], specific disease associated *CFTR* mutations [90-92], and conditional CFTR null alleles [93] have contributed to the understanding of molecular mechanisms of CF. However, mice do not recapitulate several aspects of CF lung disease pathogenesis. As discussed above, many studies evaluating integrating gene transfer vector delivery to the lungs of mice have been conducted. For these reasons, we now discuss efforts

Tuggle and colleagues used zinc finger nucleases to disrupt CFTR exon 3 in rats [94]. CFTR-/ rats recapitulate many aspects of human disease including intestinal obstruction, obstruction of the vas deferens, and abnormalities in nasal mucus production. It is currently not clear if CFTR null rats develop lung disease. To date, no gene correction studies have been reported

CFTR null ferrets were developed using AAV-mediated gene targeting in somatic cells, nuclear transfer, and cloning [95]. Unlike CF mice, CF ferrets develop early and reproducible lung infections that make it a promising platform for testing lung-directed CF therapies [95]. There

that appears to be less active in humans or ferrets [95, 98-100]. Second, in humans and ferrets, goblet cells are the predominant secretory cell type of the cartilaginous airways [101-104], whereas in mice the analogous secretory cell type is the club cell [105, 106]. Third, SMGs are virtually absent in murine cartilaginous airways, with only a handful in the most proximal regions of the trachea [106, 107]. SMGs are important for airway innate immunity in the ferret [108] and humans [109, 110], and a potentially valuable site for CFTR expression [111-114]. Lentiviral gene transfer to the wild-type neonatal ferrets using EIAV- and FIV-based vectors expressing fluorescent reporter genes was recently reported [115]. The EIAV was pseudotyped with hemagglutinin (HA) from avian influenza A virus [74] and the FIV vector was pseudo‐

channels in

transport [96, 97], a pathway

are several potential reasons for these species differences. First, Ca++-activated Cl-

the mouse airway may compensate for cAMP-mediated CFTR Cl-

**4. Animal models of cystic fibrosis**

334 Cystic Fibrosis in the Light of New Research

**4.1. CF rat**

in this novel model.

**4.2. CF ferret**

to deliver integrating vector to new animal models CF.

Pigs are an important model for many studies of human cardiovascular diseases, injury and repair, surfactants, inflammation, and pulmonary diseases (reviewed in [101]). Compared to rodents, the pig lung is anatomically and physiologically more similar to humans [117, 118] and has been studied extensively in xenotransplantation. The prenatal maturation of the pig lung is similar to humans and includes extensive alveolarization [119]. Pig airway branching and cell composition is much more akin to human airways than to those of mice. The cell types comprising the conducting airway epithelium in pigs and humans are similar, and notably lack the high percentage of club cells typical of mice. The pig bronchial epithelium is pseu‐ dostratified and contains ciliated, basal, and goblet cells, and abundant SMGs (reviewed in [101]). Importantly, the distribution of SMGs in the conducting airways and the CFTRdependent and -independent secretion of liquid and macromolecules is similar to humans [112, 120-122].

Pigs with CFTR null and ∆F508 knock-in alleles were generated by AAV-mediated homolo‐ gous recombination and somatic cell nuclear transfer [99]. Breeding heterozygous male and females generated homozygous *CFTR-/-* pigs, and their striking neonatal phenotype was described [99, 123]. Newborn CF pigs exhibit severe disease similar to humans including pancreatic insufficiency, meconium ileus with intestinal obstruction, absence of the vas deferens, and evidence of liver and gall bladder disease [123]. Importantly, CFTR null and ∆F508 pigs spontaneously develop lung disease with many features similar to humans with CF including bacterial infection, inflammation, abnormal mucociliary clearance, bronchiecta‐ sis, and remodeling.

In a recent study, we compared HIV- and FIV-based lentiviral vectors in well-differentiat‐ ed human and pig airway epithelia [66]. FIV transduced pig airway epithelia with greater efficacy than HIV, but both FIV and HIV transduced human airway epithelia with equal efficacy [66]. We further screened a number of envelope glycoproteins and identified GP64 as one of the most efficient pseudotypes for transduction and persistent expression in both pig and human epithelial cells [66]. A mCherry marker virus was delivered to wild-type pigs 4 weeks of age. A bolus dose of GP64-FIV vector was delivered to the ethmoid sinuses or to the tracheal lobe through a catheter threaded through the suction channel of a pediatric bronchoscope. We estimated the range of transduction efficiencies in the pig airways to be from <1 to 7%. In future studies, we will deliver CFTR expressing vector to CF pigs to determine the preferred gene transfer targets and the level of CFTR correction required to prevent or slow disease progression
