**7. Towards gene therapy of cystic fibrosis using a genomic construct**

Cystic Fibrosis is the most common genetic disease in the Caucasians caused by mutations in the *CFTR* gene which is 250 kb long and encodes a cAMP regulated transmembrane Clion channel in epithelial cells of several organs. The most severe implications which eventually lead to death are in the lungs (Boucher, 2002).

For several reasons including the easy access to the respiratory tract without any intervention procedures, the cloning and the characterization of the *CFTR* gene (Riordan et al., 1989; Rommens et al., 1989) and the expectation that even relatively low levels of expression of the gene may have a therapeutic outcome (Dorin et al., 1996), Cystic Fibrosis became an ideal target for gene therapy.

Fig. 3. Linking of two overlapping BACs into a single larger BAC

eventually lead to death are in the lungs (Boucher, 2002).

became an ideal target for gene therapy.

**7. Towards gene therapy of cystic fibrosis using a genomic construct** 

Cystic Fibrosis is the most common genetic disease in the Caucasians caused by mutations in the *CFTR* gene which is 250 kb long and encodes a cAMP regulated transmembrane Clion channel in epithelial cells of several organs. The most severe implications which

For several reasons including the easy access to the respiratory tract without any intervention procedures, the cloning and the characterization of the *CFTR* gene (Riordan et al., 1989; Rommens et al., 1989) and the expectation that even relatively low levels of expression of the gene may have a therapeutic outcome (Dorin et al., 1996), Cystic Fibrosis In previous preclinical and clinical studies where *CFTR* cDNA-heterologous promoter systems and different viral vectors were used for the delivery and expression of the transgene, some expression has been shown in transgenic mice and low levels of transient correction of Cl- ion transport deficiency has been observed in patients but no significant clinical improvement has been achieved (Griesenbach & Alton, 2009).

Due to the strict regulation of expression of the *CFTR* gene at specific developmental stages and in specific tissues, controlled by regulatory elements found before, after and within the gene (McCarthy & Harris, 2005), the use of constructs covering the whole genomic locus of the gene may have a better therapeutic potential for Cystic Fibrosis. To date, the only transgene that has fully restored transgenic mice, which did not express endogenous CFTR and would normally die, is the intact gene present on a YAC of approximately 300 kb in length (Manson et al., 1997). However, YAC vectors have the disadvantage of being difficult to shuttle between cells and are inherently unstable and therefore have been replaced by BACs. The *CFTR* gene is one of the large human genes that have not been found to be contained intact in any of the sequenced BACs available from the Human Genome Project. For this reason, the technology described in section 6.3 was developed and used to construct a BAC vector carrying the whole *CFTR* gene and the associated regulatory elements (Kotzamanis et al., 2009). Successful transcription of the gene to a correctly spliced mRNA has been demonstrated in a mouse cell line. This BAC is the only CFTR genomic system available on a convenient vector and may be the basis for non viral gene therapy for Cystic Fibrosis.

Viral approaches to gene therapy for Cystic Fibrosis suffer from gene delivery barriers such as absence of viral receptors in the respiratory epithelium and safety concerns such as provocation of an inflammatory response. This makes either *in vivo* or *ex vivo* non viral gene therapy an attractive avenue of research. However, several issues need to be solved before any clinical application. For instance, the *in vivo* delivery of non viral vectors is limited by the low efficiency, which becomes lower when bigger constructs are used. The demonstration that bone marrow-derived MSCs were able to differentiate to several types of cells including airway epithelial (Wang et al., 2005) indicated a potential application in an *ex vivo* approach but this is limited by poor data on how the *ex vivo* corrected MSCs can be administered and engrafted in the lung of Cystic Fibrosis patients.

Ideally, genetic manipulation with the CFTR BAC whether in the form of *in vivo* or *ex vivo* gene therapy would have to confer permanent transgene expression to avoid repeated gene or cell administration, respectively. In this regard, any of the systems that allow episomal maintenance or targeted integration at non-associated with carcinogenesis regions (described in sections 4 and 5) would have to be added to the CFTR BAC. The methodology required to add small sequences such as the *OriP*/EBNA-1 or the *S/MAR* elements, or large sequences such as the 70-kb alphoid array onto the CFTR BAC is available (see sections 6.1 and 6.2). Moreover, several methods for either *in vivo* or *ex vivo* delivery of the final construct to the respiratory epithelium have been developed and are available for use with the genomic CFTR-locus containing BAC (see section 3).
