**1. Introduction**

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (*CFTR*) gene, a cAMP-regulated anion channel. While our knowledge of CFTR function has advanced greatly since the discovery of the gene in 1989, CF remains fatal [1, 2].

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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 therapeutic strategy for a devastating disease.


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

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 systems.

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" is not nearly as simple as it appears at first glance. As such, we explore a variety of relevant outcome measures. In addition, there are challenges that need to be taken into consideration for pre-clinical and clinical *in vivo* studies, such as: delivery, efficiency, persistence, the potential for insertional mutagenesis, and cell types to transduce.
