# Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies
## 4 Gene Therapy for CF
### Targeting ENaC  
Since the CFTR gene mutation causes hyperactivation of ENaC, the absorption of Na\({}^{+}\) ions is enhanced and the lung airway mucus becomes dehydrated. A previous report revealed that the in vivo overexpression of ENaC in mice lungs resulted in increased absorption of Na\({}^{+}\) ions and reduction in ASL [161]. Therefore, the inhibition of ENaC expression is a promising therapeutic approach for the treatment of CF. One method for the inhibiting the expression of ENaC-encoding genes (_SCNNIA_, _SCNNIB_, _SCNNIG_, and _SCNNID_ encoding \(\alpha\), \(\beta\), \(\gamma\), and \(\delta\) ENaC subunits, respectively) involves the use of a single strand nucleic acid known as antisense oligonucleotide (ASO). When this oligonucleotide is hybridized to mRNA, RNase H is triggered to slice the hybridized mRNA [162]. Targeting the \(\alpha\)-subunit of ENaC in the lung, using ASO, could inhibit the cationic channel activity [163; 164; 165]. Another study showed the ability of aerosolized ENaC ASO containing wing modifications to inhibit ENaC mRNA in CF-like mice models [148]. This aerosolized ENaC ASO helped cure CF symptoms, like airway hyper-responsiveness and inflammation, and has reached the phase I clinical trial [165]. Another strategy to inhibit ENaC expression is using small interference RNA (siRNA). siRNA is a double stranded RNA, of approximately 21 base pair (bp) RNA nucleotides, consisting of a passenger strand that is identical to a part of the targeted mRNA and a guided strand that is complement to a part of the target mRNA in an open reading frame. When siRNA is taken up by a cell, it removes the passenger strand during the activation of the RNA-induced silencing complex (RISC) [166]. The guided strand is led by the RISC, which contains four Argonaute subunits (Ago 1-4), to its target mRNA, where it is cleaved by Ago 2 [167; 168; 166]. The primary advantage of gene expression inhibition using siRNA is that the guided strand, presented in RISC, is protected from cytoplasmic endonucleases [169; 170]. Additionally, the RISC-incorporated guided strand triggers the cleavage of multiple mRNA targets expressing the same protein [171]. siRNA has also been used to evaluate the genes responsible for the regulation of ENaC expression; the inhibition of diacylglycerol kinase iota protein, which is involved  
Figure 4: Gene-based therapeutic approaches for targeting faulty CFTR using different methods: (**a**) antisense oligonucleotide, (**b**) gene editing, and (**c**) gene replacement. in the phosphatidylinositol biphosphate metabolism, downregulates ENaC function, leading to the normal absorption of Na\({}^{+}\) ions and fluid in CF airways [172]. The direct inhibition of ENaC by siRNA has been evaluated both in vitro and in vivo [173; 174; 175; 34; 176; 177]. In the in vitro evaluation, the levels of mRNA expressing the \(\alpha\), \(\beta\), and \(\gamma\) subunits of ENaC were measured in untreated primary bronchial epithelial cells. The results showed that the \(\alpha\) subunit was more abundant compared to \(\beta\) and \(\gamma\) subunits [175]. Following siRNA treatment, a reduction was observed in the absorption of Na\({}^{+}\) ions and fluid, which persisted for over seven days [158]. Another in vitro study showed that a significant increase in ASL was observed only when both the \(\alpha\) and \(\beta\) subunits of ENaC were knocked down simultaneously using siRNA [34], while the siRNA-mediated knockdown of the \(\alpha\) subunit mRNA alone reduced the function of ENaC [174]. In vivo knockdown of the \(\alpha\) subunit was successfully performed using a cationic liposome carrier containing a peptide ligand [173]. Additionally, it was observed that repeated in vivo doses of this siRNA-liposome lipoplex improved the knockdown of the \(\alpha\) subunit of ENaC mRNA [176]. Furthermore, the in vitro translocation of this formulation across the mucus membrane demonstrated its ability to cross the mucus layer faster than the naked siRNA [176].