**5. Outcome measures**

In pre-clinical studies of CF gene therapy, it is vital to define metrics of correction before the studies are initiated. It would be naïve to simply deliver vector to the airways and "look and see" if the airway disease is cured. CF is a complex disease with many phenotypic features and clearly defining the disease progression in an untreated CF animal model is vital. As experience is gained with new animal models, additional assays for correction will be established and refined. Importantly, these metrics may apply to multiple gene correction but may not be feasible in all animal models.

### **5.1. Quantitative real-time PCR and CFTR protein expression**

Quantitative real-time RT-PCR is a sensitive assay for measuring vector expressed *CFTR* mRNA [123]. At progressive time-points post vector delivery, whole tissue or brushings of nasal or tracheobronchial epithelia can be obtained. Silent mutations can be engineered into the vector expressed *CFTR* cDNA so that transgene expression can be differentiated from endogenous *CFTR*. Using a similar strategy, vector genome copy number can be estimated. Genomic DNA from a portion of the same tissue or epithelial brushings can be purified and copy number estimated by normalizing to endogenous DNA and a standard curve. Appro‐ priate controls would include wild type and untreated affected littermates. To identify cells expressing the CFTR protein, immunohistochemistry and immunofluorescence protocols have been reported [123]. Using these approaches, the percentage of cells expressing CFTR and the cell types expressing CFTR can be determined [124].

### **5.2. Functional correction**

Nasal potential difference (NPD) is an established assay for demonstrating *in vivo* correction of CFTR-dependent Cl transport [125-127]. For many of these studies, vector was delivered nasally and the NPD was used as evidence of CFTR complementation. Ideally, for integrating vectors, predelivery, early post-delivery, and late post-delivery timepoints in the intrapulmo‐ nary airways should be measured. As early as 1 week after gene transfer, the nasal voltage and its response to amiloride, low Cl- , and cAMP agonists can be measured [90, 123, 128]. Animals could be followed with serial monthly nasal voltage measurements over a 12-month or longer period to document persistence of expression. Importantly, CFTR is an anion channel that conducts both Cl and HCO3 - . As mentioned above, correcting ~10% of cells is often cited as a benchmark for restoring Cl- transport and correcting the clinical phenotype. However, other studies also suggest that defective HCO3 transport might be relevant to disease. Multiple studies of CF mouse cervical mucus [46, 47], CF mouse small intestinal mucus [48], and human CF nasal SMGs [49] support the importance of CFTR-dependent HCO3 transport in CF pathogenesis. Loss of CFTR-dependent HCO3 - transport acidifies liquid produced by surface epithelia [129, 130] and secretions from SMGs [49]. Thus, measurements of HCO3 transport may also be an important metric of functional correction.

As a result of abnormal CFTR-dependent HCO3 secretion, airway surface liquid pH is acidified. The ASL of primary cultures of CF pig airway epithelia [130], newborn CF pig airways [130], and the nasal pH of newborn babies with CF [131] is acidic. In CF pigs, the acidity has been shown to impair bacterial killing [130]. In addition, there are new techniques to measure mucus viscosity, mucociliary clearance (MCT), and lung function in large animal models [101]. Importantly, defects in airway MCT and SMG mucus detachment recently were identified in CF [132]. The bacterial killing defect is a quantifiable characteristic of CF airways. Bacterial killing is impaired as a result of reduced bicarbonate anion secretion and increasing ASL pH rescues bacterial killing [130].

### **5.3. Reduced infection and inflammation**

A goal of gene therapy for CF is to prevent the onset or reduce the progression of lung disease. Signs of reduced infection and inflammation in treated animals can be visually inspected in the airways. Bronchoscopy can be used to detect signs of inflammation, such as mucosal inflammation and excessive purulent secretions. Total cell counts, cell differentials, and cytokine levels are obtained from bronchoalveolar lavage (BAL) as standard assay for infection and inflammation. In addition, standard quantitative microbiologic techniques are used to identify and quantify BAL bacteria [133]. Biopsies can also be obtained from larger animal models such as the CF pig. These samples can also be used for sequencing-based analyses. As we learn more about the disease progression in new animal models of CF, improved metrics of functional correction are being developed. High-resolution computerized tomography (HRCT) facilitates detailed structural analysis of the airways [101]. HRCT scans can discern anatomic changes in the airways over time in control and treated animals [134].
