**2. Correcting the appropriate cells**

While CF is a multi-organ system disease, most people with CF die of progressive lung disease that begins early in childhood and is characterized by chronic bacterial infection and inflam‐ mation [2]. Nearly 90% of CF patients have at least one copy of the ΔF508 mutation, but there are >2,000 disease causing mutations that result in a range of disease severities [2]. These mutations can be divided into six classes based on the type and consequence of the mutation (Table 1): class I, no synthesis; class II, defective processing; class III, defective regulation; class IV, altered conductance; class V, reduced synthesis; and class VI, accelerated turnover [3]. However, new mutations continue to be identified and one mutation may fit into more than one category by disrupting CFTR transcription, protein trafficking, or protein regulation in more than one way. Pharmacologic approaches aimed at activating alternative ion transport pathways [4-7], reducing inflammation [8, 9], and inhibiting or eliminating bacterial infection [10, 11] are active areas of therapeutic development. There is also intense interest in identifying interventions that might restore function to the mutant protein [5, 6, 12, 13]. The promise of restoring function to mutant protein was recently validated in a clinical trial for the CFTR conductance mutation G551D present in 2%–3% of CF patients [6]. However, unlike small molecule potentiators or correctors, a CFTR gene replacement approach would be efficacious regardless of the disease causing mutation and is potentially a single dose, life-long curative

**Class ~Frequency Mutation Type Common Representative CFTR Protein Outcome**

**II** 70% Missense ∆F508 Defective Processing **III** 2%–3% Missense G511D Defective Regulation **IV** <2% Missense R117H Altered Conductance **V** <1% Missense, splice 3349+10KB Reduced Synthesis **VI** <1% Missense N287Y Accelerated Turnover

There are a great number of vector options for *CFTR* gene delivery. Non-integrating viral vectors (i.e., adenovirus or adeno-associated virus) and non-integrating nonviral vectors (i.e., plasmid DNA or *in vitro* transcribed RNA) each have important attributes and have resulted in significant advances in the CF gene therapy field (reviewed in [16-18]). However, potential limitations to these episomal expression systems may include gradual decreases in transgene expression over time and limiting host immune responses following vector readministration. These pitfalls could be avoided if a therapeutic transgene is stably integrated into the genome of a progenitor cell population. Thus, in this chapter, we focus on the use of integrating vectors for gene delivery, although some of the topics covered will be relevant to multiple vector

This chapter discusses the common and emerging options for integrating vectors and efforts to deliver integrating vectors to CF animal models. Defining "curing CF with gene therapy"

**I** 10% Nonsense, splice G542X No CFTR

**Table 1.** Six classes of mutations in CFTR that result in CF disease [3, 14, 15].

systems.

therapeutic strategy for a devastating disease.

328 Cystic Fibrosis in the Light of New Research

Because pulmonary disease is generally the most life-limiting complication of CF, gene therapy strategies focus on lung delivery of CFTR. Regardless of the gene delivery tool, an important consideration for CF gene therapy is the target cell. In the proximal airways, CFTR is normally most abundant in surface epithelial cells including ciliated cells, surface columnar cells, and submucosal gland epithelia (SMGs) [19]; in distal airways only superficial epithelia express CFTR. With this information in mind, we face two important questions. 1) What cell types need to be transduced to attain lasting expression? 2) What percentage of cells needs to be trans‐ duced to correct CF lung disease? A goal of gene transfer to the pulmonary epithelium with integrating vectors is to correct the CFTR defect in a population of cells that could pass the corrected gene to their progeny, thus eliminating the need for vector readministration. There appear to be several epithelial cell types in the lung that provide these functions, which has led to controversy regarding which cells to target for CF gene therapy. Arguments can be made in support of the necessity to correct basal cells [20, 21] and non-ciliated columnar cells of the airways [22-24], SMGs [25-27], club cells [28, 29], and alveolar type II cells [30, 31] in the distal lung.

Compelling evidence from both *in vitro* and *in vivo* studies indicate that basal cells are multipotent proximal airway progenitor cells that repopulate pulmonary epithelia under normal conditions and during regeneration (reviewed in [32-34]). Cell-labeling experiments with transgenic mice show that basal cells give rise to labeled basal, ciliated, and club cells, thus fulfilling the definition of progenitor cells [35, 36]. Several studies suggest that basal cells from human trachea or bronchi will repopulate denuded tracheal xenografts or differentiated epithelial cells *in vitro* [37-40]. Hematopoietic stem cells are an example showing that a single stem cell type can reconstitute a whole organ; however, there is no convincing evidence that a multipotent airway stem cell is capable of replenishing all regions of the intrapulmonary epithelium. The current literature supports that tracheal, bronchiolar, and alveolar epithelia are maintained by regionally distinct progenitor cell lineages.

What percentage of cells needs to be transduced to functionally correct the CF phenotype *in vivo*? This is one of the most important questions in the field of CF gene therapy, but remains unanswered. At least five studies examined the relationship between percentage of cells expressing CFTR and transepithelial Cl secretion [41-45]. With relatively good agreement, they suggest that expressing CFTR in 5%–15% of cells restores Cl secretion to near wild-type levels. As such, the benchmark of correcting ~10% of the cells is often cited. However, there are many caveats to this number. Indeed one limitation is that many of these studies were performed using *in vitro* models. In addition, as discussed below, other studies suggest that defective HCO3 transport through CFTR might be more relevant to early disease pathogenesis than Cl secretion [46-49]. The relationships between HCO3 secretion, airway surface liquid pH, bacterial killing, mucociliary clearance, and mucus viscosity may be as important as Clsecretion as metrics for disease correction. The short answer to the question posed above is "we do not know"; however, given current animal models and improved vector technologies, the experiments necessary to address the question are feasible. Indeed, as we discuss below, existing vector technologies are being optimized for lung gene transfer and novel integrating vectors are being engineered.
