**4. Genetic medicines**

Traditional CF care has focused upon the management of the systems affected in individuals with CF. However, the identification of the CFTR gene enabled researchers to focus on treatments strategies, which could address the underlying genetic defect*.* The major cause of morbidity and mortality in CF is secondary to lung disease. Hence, if abnormal CFTR in the lungs could be replaced with wt-CFTR during the neonatal period, prior to parenchymal lung damage or bacterial colonization, morbidity and mortality could be significantly altered within the CF population [24]. Various approaches have been investigated within the field of "genetic medicines" and unfortunately to date none are a viable treatment option outside of clinical trials.

"Genetic medicines" comprise of four different treatment approaches:


The potential benefit of these therapies is that theoretically they should be suitable for the treatment of all individuals with CF, regardless of genotype. Currently gene therapy has made the greatest advancement towards being a clinical treatment and so the main focus of this section will be around gene therapy.

As the respiratory system is so central to CF disease and because initial thoughts were that gene therapy targeting the lungs would be easy to deliver, locally directed gene therapy to the respiratory epithelium was the method of choice. Furthermore, gene therapy can complement any CFTR causing mutation. However, for such treatment to be successful various issues had to be addressed, including the choice of delivery vector, method of delivery to the airways, translocation of the genetic information and ultimately ensuring that there was appropriate expression of the normalized CFTR gene [25]. These various issues will each be discussed to provide insight in the difficulties experienced in trying to develop "genetic medicines."

The lungs are comprised of terminally differentiated epithelial cells, which are slowly replaced by stem/progenitor cells. Any form of gene therapy must be able to be either repeatedly delivered to the terminally-differentiated cell surface or be able to alter the stem/progenitor cells within the lungs. However, the lung has evolved physical and immune mechanisms to protect against pathogens and particulate materials, which impacts upon choice of vector delivery [26, 27].

Delivery vectors are largely either viral or non-viral in nature with viral ones felt to be more efficient. This is because they have evolved to overcome the barrier mechanisms present within the lungs. Adenoviruses (Ad) and adeno-associated viruses (AAV) have a natural trophism for the lungs, are DNA-based and thus were the initial choices to study. Adenoviruses are small in size and thus to insert the CFTR DNA correctly within the adenoviral genome, viral DNA must be removed, impacting upon the viral cytopathic effect. These vectors were found to have poor efficacy due to the pre-existing and induced immune responses, and thus cannot be repeatedly administered as required for these treatments because of the short life span of bronchial epithelial cells.

Other viral vectors that have been investigated are recombinant lentivirus (rLV). These agents are RNA-based and can integrate into the genome. This can be advantageous as it ensures that the vector is passed down the cell lines during division but it also does have the risk of inducing insertional mutagenesis [21, 26, 28]. However, ultimately other vectors were needed to be formulated, ones which had a minimal risk of immunogenicity and thus could be repeatedly administered.

Non-viral gene transfer agents complexed to plasmid DNA were therefore developed [21, 29]. These have been more successful than their viral vector counterparts

**43**

*Overview of CFTR Modulators and Gene Therapy DOI: http://dx.doi.org/10.5772/intechopen.91022*

these options being viable in the clinical setting.

quality of life measures [30].

**5. CFTR modulator agents**

channel; and

**5.1 CFTR modulator drug design**

developed;

and have been investigated in Phase IIb studies. Patients who were 12 years and older were treated with the non-viral CFTR gene-liposomal complex pGM169/ GLG7A as a nebulized therapy over a one-year period. The repeated nebulization each month resulted in a reduction in the progression of CF lung disease by a modest amount when compared with placebo. The percentage change in the forced expiratory volume in 1 second (FEV1) over 12 months was −0.4% versus −4.0% in the placebo arm. Hence, although no improvement in lung function was seen, this study was promising as rate of lung function decline does impact morbidity and mortality in CF. However, disappointingly also there were no improvements in

As described in the above study the agents utilized were delivered via inhalation methods. This has been found to be the easiest method for repeated treatment applications. However, difficulties have arisen ensuring adequate lung deposition of drug, related to particle size and the type of nebulisers used. Additionally, any aerolised drug delivered must retain its biological function post-delivery [31, 32]. Other strategies for ensuring corrected CFTR protein production is through

mRNA therapy and mRNA repair as described above. The benefit of these approaches is that they do not require translocation of the therapy across the nuclear membrane. Nanoparticle-chemically modified mRNA has resulted in lung function improvements in animal models without any immune reactions despite repeated applications. Also, there is evidence that these therapies can restore chloride channel activity [33, 34]. Ongoing work and investigation are required prior to

CFTR modulator agents are small molecules which 'modulate' the function of the abnormal CFTR protein. Unlike gene therapy, they do not alter the CFTR gene. However, these agents do manipulate the underlying genetic consequence of CF mutations. Currently two different classes of modulator agents have been

i.potentiators which 'potentiate' the cAMP-mediated gating of the CFTR

High-throughput drug discovery programs enabled the development of such agents. These discovery programs were established to identify active compounds ("hits") from large chemical libraries suitable for industrial-scale screening. High-throughput screening (HTS) assays need to be robust, have high throughput using small sample volumes together with adequate sensitivity, reproducibility and accuracy to ensure differentiation between a very large amount of compounds [35]. Ion channels are key targets for drug design and thus HTS have been an important

The two classes of small molecules for CFTR protein modulation were identified via HTS techniques from libraries that consisted of chemically diverse drug-like and lead-like compounds acquired from both commercial vendors and internal medicine chemistry programs. If compounds had an activity >2.5 standard deviations (SD) from the mean, then they received further testing. For example, from

ii. correctors which 'correct' defects in protein trafficking.

part of such drug discovery processes, including for CF [36, 37].

#### *Overview of CFTR Modulators and Gene Therapy DOI: http://dx.doi.org/10.5772/intechopen.91022*

*Cystic Fibrosis - Heterogeneity and Personalized Treatment*

production of normal CFTR protein;

leading to recombination occurring;

the CFTR mRNA [21].

span of bronchial epithelial cells.

"Genetic medicines" comprise of four different treatment approaches:

resulting in the production of normal CFTR protein;

and so the main focus of this section will be around gene therapy.

materials, which impacts upon choice of vector delivery [26, 27].

risk of immunogenicity and thus could be repeatedly administered.

Delivery vectors are largely either viral or non-viral in nature with viral ones felt to be more efficient. This is because they have evolved to overcome the barrier mechanisms present within the lungs. Adenoviruses (Ad) and adeno-associated viruses (AAV) have a natural trophism for the lungs, are DNA-based and thus were the initial choices to study. Adenoviruses are small in size and thus to insert the CFTR DNA correctly within the adenoviral genome, viral DNA must be removed, impacting upon the viral cytopathic effect. These vectors were found to have poor efficacy due to the pre-existing and induced immune responses, and thus cannot be repeatedly administered as required for these treatments because of the short life

Other viral vectors that have been investigated are recombinant lentivirus (rLV). These agents are RNA-based and can integrate into the genome. This can be advantageous as it ensures that the vector is passed down the cell lines during division but it also does have the risk of inducing insertional mutagenesis [21, 26, 28]. However, ultimately other vectors were needed to be formulated, ones which had a minimal

Non-viral gene transfer agents complexed to plasmid DNA were therefore developed [21, 29]. These have been more successful than their viral vector counterparts

i.**Gene therapy:** the delivery of wt-CFTR to the cell nucleus resulting in the

ii.**mRNA therapy:** the delivery of correct CFTR mRNA to the cytoplasm

iii.**Gene editing:** repair of the mutant CFTR DNA with normal CFTR protein resulting. This requires wt-CFTR DNA to be delivered to the nucleus together with mRNA encoding a nuclease that causes a break in the DNA

iv.**mRNA editing:** CFTR mRNA delivery to the cytoplasm leading to repair of

The potential benefit of these therapies is that theoretically they should be suitable for the treatment of all individuals with CF, regardless of genotype. Currently gene therapy has made the greatest advancement towards being a clinical treatment

As the respiratory system is so central to CF disease and because initial thoughts were that gene therapy targeting the lungs would be easy to deliver, locally directed gene therapy to the respiratory epithelium was the method of choice. Furthermore, gene therapy can complement any CFTR causing mutation. However, for such treatment to be successful various issues had to be addressed, including the choice of delivery vector, method of delivery to the airways, translocation of the genetic information and ultimately ensuring that there was appropriate expression of the normalized CFTR gene [25]. These various issues will each be discussed to provide insight in the difficulties experienced in trying to develop "genetic medicines." The lungs are comprised of terminally differentiated epithelial cells, which are slowly replaced by stem/progenitor cells. Any form of gene therapy must be able to be either repeatedly delivered to the terminally-differentiated cell surface or be able to alter the stem/progenitor cells within the lungs. However, the lung has evolved physical and immune mechanisms to protect against pathogens and particulate

**42**

and have been investigated in Phase IIb studies. Patients who were 12 years and older were treated with the non-viral CFTR gene-liposomal complex pGM169/ GLG7A as a nebulized therapy over a one-year period. The repeated nebulization each month resulted in a reduction in the progression of CF lung disease by a modest amount when compared with placebo. The percentage change in the forced expiratory volume in 1 second (FEV1) over 12 months was −0.4% versus −4.0% in the placebo arm. Hence, although no improvement in lung function was seen, this study was promising as rate of lung function decline does impact morbidity and mortality in CF. However, disappointingly also there were no improvements in quality of life measures [30].

As described in the above study the agents utilized were delivered via inhalation methods. This has been found to be the easiest method for repeated treatment applications. However, difficulties have arisen ensuring adequate lung deposition of drug, related to particle size and the type of nebulisers used. Additionally, any aerolised drug delivered must retain its biological function post-delivery [31, 32].

Other strategies for ensuring corrected CFTR protein production is through mRNA therapy and mRNA repair as described above. The benefit of these approaches is that they do not require translocation of the therapy across the nuclear membrane. Nanoparticle-chemically modified mRNA has resulted in lung function improvements in animal models without any immune reactions despite repeated applications. Also, there is evidence that these therapies can restore chloride channel activity [33, 34]. Ongoing work and investigation are required prior to these options being viable in the clinical setting.
