**2. Pathophysiology of cystic fibrosis**

#### **2.1 CFTR protein structure**

CFTR codes for a complex protein, which is present in every nucleated cell of the body, however it is normally concentrated on the apical membrane of epithelial cells, primarily within the glandular epithelia. High expression of this apical anion channel is seen within the lungs, pancreas, gastrointestinal tract, vas deferens and sweat glands; reflecting the main organs affected in CF [6].

The CFTR protein is a large, unique member of the subclass C family of the ATP binding cassette (ABC) transporter proteins, which functions as an ion channel rather than an active transporter protein [7–9]. It consists of two membranespanning domains (MBDs) that form the ion channel. These domains are both connected to two cytoplasmic nucleotide-binding domains (NBD1 and NBD2), which function to gate the channel. This conformation of two MBDs and two NBDs that hydrolyse ATP are typical for most ABC transporters. However, CFTR has an additional cytoplasmic regulatory domain (R domain), inserted between NBD1 and MSD2 linking the two transporter domains. Phosphorylation of the R domain by protein kinase A enables channel opening to occur and channel activity is increased upon phosphorylation. Once phosphorylation has taken place, ATP binds to the NBDs resulting in the two NBDs forming tightly interacting dimers, which gates the channel. These movements are transmitted to the MBDs causing the ion pore to open. Channel closure results from ATP hydrolysis. The exact mechanisms underlying the regulation of the R domain and ATP-dependent gating are still not completely understood [10, 11].

#### **2.2 CFTR protein synthesis**

CFTR protein synthesis is a complex process, in part related to the size of the functional protein. As with all protein synthesis, transcription of the CFTR DNA takes place within the nucleus to create the messenger RNA (mRNA), which is transported across the nuclear membrane to the cytosolic ribosomes. There the initiation of translation occurs to create a 1480-amino acid polypeptide chain based upon the genetic code. Initially a 135- to 140-kDa core-glycosylated precursor is produced (immature CFTR). CFTR biosynthesis then proceeds through the endoplasmic

**39**

**Figure 1.**

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

translational modifications take place (**Figure 1**).

reticulum (ER) followed by the Golgi apparatus to a mature 150- to 160-kDa CFTR form which has undergone conformational folding [12]. During the secretory pathway through the ER to the Golgi and then on to the cell membrane various post-

The maturation process to create the final relatively compact CFTR protein structure is inefficient and slow. Less than 30% of newly synthesized wild-type (wt) immature CFTR molecules develop into mature CFTR proteins. For folding of the polypeptide chain to occur chaperones are required, in particular the 70 k-Da heat shock proteins (HSP70) and calnexin. In cells of individuals with the Phe508del-CFTR mutation, almost all immature molecules fail to reach final maturity and thus are degraded. This is the due to the quality control mechanisms in place within the ER, specific signals and distinct processes exist that recruit misfolded proteins to the ER-associated degradation as a final endpoint. These proteins are then directed for

Certain steps within the CFTR biosynthesis pathway are still unknown, however, data does support each domain folding independently. The native structure develops through a co-translational mechanism, possibly together with posttranslational processes that take place to create the compactly folded domains. Domain-domain interactions are key in the creation of conformationally correct CFTR [15]. Furthermore, it appears that CFTR is more sensitive to mutations in NBD1 compared with homologous mutations in NBD2, leading to issues with the conformational maturation of the whole CFTR protein. For example, the deletion of the Phe508 does not appear to grossly alter the structure of NBD1 but subsequent issues arise during the maturation process, possibly through the disruption of the interaction between NBD1 and NBD2 and despite each domain folding independently. Maturation thus requires precise folding of each domain together with the

*Cell biology of CFTR - abnormal CFTR protein results in the uncoupling of CFTR dependent processes at all levels from intracellular dynamics to cell membrane function. Reproduced with permission of the © ERS 2020: European Respiratory Journal; DOI: 10.1183/13993003.02443-2019: Accepted for publication and in press.*

degradation via the ubiquitin-proteasome pathway [12–14] (**Figure 1**).

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

*Cystic Fibrosis - Heterogeneity and Personalized Treatment*

**2. Pathophysiology of cystic fibrosis**

sweat glands; reflecting the main organs affected in CF [6].

**2.1 CFTR protein structure**

completely understood [10, 11].

**2.2 CFTR protein synthesis**

Due to the multi-system nature of the disease, treatment has classically focused on therapies and systems of care that aim to improve salt and fluid balance and nutritional status, alongside reducing airway inflammation and lung parenchymal destruction. These multi-disciplinary management approaches have been instrumental in the improvements seen in life expectancy. The median predicated survival of an individual born today with CF is 47 years, compared with 20 years at the time when CFTR was discovered in 1989 [2]. However, to have a true impact upon the management of these patients and to alter the disease trajectory, treatment options needed to also include approaches targeting the underlying genetic mutation. This chapter will include a review of the structure of the CFTR protein, its biosynthesis and the pathophysiology of CF so as to provide a basis from which to discuss the various therapeutic strategies that have more recently been developed for modulating CFTR protein function. Also, a discussion regarding gene therapy will be included so as to enable contrasts and comparisons to be made between the different therapies being evolved to address the underlying genetic defect in CF

CFTR codes for a complex protein, which is present in every nucleated cell of the body, however it is normally concentrated on the apical membrane of epithelial cells, primarily within the glandular epithelia. High expression of this apical anion channel is seen within the lungs, pancreas, gastrointestinal tract, vas deferens and

The CFTR protein is a large, unique member of the subclass C family of the ATP binding cassette (ABC) transporter proteins, which functions as an ion channel rather than an active transporter protein [7–9]. It consists of two membranespanning domains (MBDs) that form the ion channel. These domains are both connected to two cytoplasmic nucleotide-binding domains (NBD1 and NBD2), which function to gate the channel. This conformation of two MBDs and two NBDs that hydrolyse ATP are typical for most ABC transporters. However, CFTR has an additional cytoplasmic regulatory domain (R domain), inserted between NBD1 and MSD2 linking the two transporter domains. Phosphorylation of the R domain by protein kinase A enables channel opening to occur and channel activity is increased upon phosphorylation. Once phosphorylation has taken place, ATP binds to the NBDs resulting in the two NBDs forming tightly interacting dimers, which gates the channel. These movements are transmitted to the MBDs causing the ion pore to open. Channel closure results from ATP hydrolysis. The exact mechanisms underlying the regulation of the R domain and ATP-dependent gating are still not

CFTR protein synthesis is a complex process, in part related to the size of the functional protein. As with all protein synthesis, transcription of the CFTR DNA takes place within the nucleus to create the messenger RNA (mRNA), which is transported across the nuclear membrane to the cytosolic ribosomes. There the initiation of translation occurs to create a 1480-amino acid polypeptide chain based upon the genetic code. Initially a 135- to 140-kDa core-glycosylated precursor is produced (immature CFTR). CFTR biosynthesis then proceeds through the endoplasmic

**38**

patients.

reticulum (ER) followed by the Golgi apparatus to a mature 150- to 160-kDa CFTR form which has undergone conformational folding [12]. During the secretory pathway through the ER to the Golgi and then on to the cell membrane various posttranslational modifications take place (**Figure 1**).

The maturation process to create the final relatively compact CFTR protein structure is inefficient and slow. Less than 30% of newly synthesized wild-type (wt) immature CFTR molecules develop into mature CFTR proteins. For folding of the polypeptide chain to occur chaperones are required, in particular the 70 k-Da heat shock proteins (HSP70) and calnexin. In cells of individuals with the Phe508del-CFTR mutation, almost all immature molecules fail to reach final maturity and thus are degraded. This is the due to the quality control mechanisms in place within the ER, specific signals and distinct processes exist that recruit misfolded proteins to the ER-associated degradation as a final endpoint. These proteins are then directed for degradation via the ubiquitin-proteasome pathway [12–14] (**Figure 1**).

Certain steps within the CFTR biosynthesis pathway are still unknown, however, data does support each domain folding independently. The native structure develops through a co-translational mechanism, possibly together with posttranslational processes that take place to create the compactly folded domains. Domain-domain interactions are key in the creation of conformationally correct CFTR [15]. Furthermore, it appears that CFTR is more sensitive to mutations in NBD1 compared with homologous mutations in NBD2, leading to issues with the conformational maturation of the whole CFTR protein. For example, the deletion of the Phe508 does not appear to grossly alter the structure of NBD1 but subsequent issues arise during the maturation process, possibly through the disruption of the interaction between NBD1 and NBD2 and despite each domain folding independently. Maturation thus requires precise folding of each domain together with the

#### **Figure 1.**

*Cell biology of CFTR - abnormal CFTR protein results in the uncoupling of CFTR dependent processes at all levels from intracellular dynamics to cell membrane function. Reproduced with permission of the © ERS 2020: European Respiratory Journal; DOI: 10.1183/13993003.02443-2019: Accepted for publication and in press.*

correct inter-domain assembly to create a stable structure that will not be submitted to ER-associated degradation [13, 16].

If the protein passes through all the checkpoint steps within the ER, it can exit and be transported through the Golgi apparatus in vesicles where the removal and addition of new glycan units takes place, increasing the molecular size of CFTR. It is becoming clear that some wt-CFTR might bypass these processes in the delivery pathway to the plasma membrane. Once at the membrane, levels of CFTR vary depending upon the balance of anterograde trafficking, endocytosis and recycling. Recycling of internalized CFTR to the plasma membrane is thought to assist with sustaining a functional pool of CFTR at the membrane level [15].

#### **2.3 Pathophysiology**

CFTR functions as a chloride and bicarbonate channel. Loss of functional CFTR proteins result in reduced chloride efflux from epithelial cells leading to depletion of the cell surface fluid and altering its pH and osmolarity. CFTR also regulates the activity of various other key processes within the cell, including the activity of other ion channels, such as the sodium epithelial channel (ENaC; the amiloride-sensitive sodium channel). Suppressed CFTR activity can lead to unopposed reabsorption of sodium and water via ENaC, causing additional dehydration of the cell surface layer [6]. Mucociliary clearance is further delayed due to abnormally adherent mucus. Dysfunctional CFTR also impacts upon mitochondrial function, the innate immunity and dysregulates inflammation [17–19]. Within the airways, this results in an environment that is susceptible to unchecked inflammation and chronic bacterial infection.

Although multiple processes both intra- and extra-cellularly are altered by dysfunctional CFTR proteins, chloride transport at the cell surface is generally considered to be the major driver of the pathophysiological disease. Functional chloride channel changes are thus likely to represent an easily accessible surrogate marker of all processes affected in CF, with sweat chloride testing being relatively easy to perform. In vitro studies have shown that only 6–10% of residual CFTR function is required to restore chloride transporting properties seen in 100% correct cells, with cell-cell coupling providing a means of amplification of the functional properties [20]. Individuals with CF who have approximately 10% CTFR expression per cell do not generally develop lung disease or the full range of classical CF disease. To date, it is unclear whether low level expression (10%) of CFTR in all cells is comparable to 10% of CFTR cells with full correction [21]. In addition, even individuals with a single CFTR mutation may have organ dysfunction in the context of a second "hit" such as smoking [22].

The general identification of mutations in the structure of CFTR has been centralized for clinician reference. CFTR2.org identifies over 2000 variants of the protein, of which over 400 are disease-associated. The majority of variants are rare and not confirmed to be disease-associated, however the large number of variants indicates the lack of stability of the CFTR gene in population dynamics [1].

#### **3. Classification of CFTR mutations**

Classification systems of common CFTR mutations have been developed to assist with understanding of the consequential molecular defect. The established classification system includes six different classes (**Figure 2**). Different mutations can result in no functional protein production, impaired protein trafficking, altered channel gating, decreased channel conductance, reduced protein synthesis and decreased protein stability [6]. Each class confers a different disease severity, which

**41**

functional protein [23].

**Figure 2.**

**4. Genetic medicines**

outside of clinical trials.

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

is related to the degree of CFTR dysfunction and has prognostic implications for patients. However, each mutation may have features of more than just one class. For example, Phe508del is predominately a class II mutation but also has both class III and class VI properties. More recently, other classification systems have been proposed, which subdivide class I mutations (no functional CFTR protein) into two groups so as to take into account whether the mutation leads to no mRNA or no

*CFTR classification table. The classification systems divide mutations into discrete groups determined by the predominant CFTR defect. However, these systems may not be mutually exclusive for all mutations. For example, the p.Phe508del-CFTR is predominately class II but does also have some class III and class VI properties. Reproduced with permission of the © ERS 2020: European Respiratory Journal; DOI:* 

*10.1183/13993003.02443-2019: Accepted for publication and in press.*

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

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


#### **Figure 2.**

*Cystic Fibrosis - Heterogeneity and Personalized Treatment*

to ER-associated degradation [13, 16].

**2.3 Pathophysiology**

such as smoking [22].

**3. Classification of CFTR mutations**

correct inter-domain assembly to create a stable structure that will not be submitted

If the protein passes through all the checkpoint steps within the ER, it can exit and be transported through the Golgi apparatus in vesicles where the removal and addition of new glycan units takes place, increasing the molecular size of CFTR. It is becoming clear that some wt-CFTR might bypass these processes in the delivery pathway to the plasma membrane. Once at the membrane, levels of CFTR vary depending upon the balance of anterograde trafficking, endocytosis and recycling. Recycling of internalized CFTR to the plasma membrane is thought to assist with

CFTR functions as a chloride and bicarbonate channel. Loss of functional CFTR proteins result in reduced chloride efflux from epithelial cells leading to depletion of the cell surface fluid and altering its pH and osmolarity. CFTR also regulates the activity of various other key processes within the cell, including the activity of other ion channels, such as the sodium epithelial channel (ENaC; the amiloride-sensitive sodium channel). Suppressed CFTR activity can lead to unopposed reabsorption of sodium and water via ENaC, causing additional dehydration of the cell surface layer [6]. Mucociliary clearance is further delayed due to abnormally adherent mucus. Dysfunctional CFTR also impacts upon mitochondrial function, the innate immunity and dysregulates inflammation [17–19]. Within the airways, this results in an environment that is susceptible to unchecked inflammation and chronic bacterial infection. Although multiple processes both intra- and extra-cellularly are altered by dysfunctional CFTR proteins, chloride transport at the cell surface is generally considered to be the major driver of the pathophysiological disease. Functional chloride channel changes are thus likely to represent an easily accessible surrogate marker of all processes affected in CF, with sweat chloride testing being relatively easy to perform. In vitro studies have shown that only 6–10% of residual CFTR function is required to restore chloride transporting properties seen in 100% correct cells, with cell-cell coupling providing a means of amplification of the functional properties [20]. Individuals with CF who have approximately 10% CTFR expression per cell do not generally develop lung disease or the full range of classical CF disease. To date, it is unclear whether low level expression (10%) of CFTR in all cells is comparable to 10% of CFTR cells with full correction [21]. In addition, even individuals with a single CFTR mutation may have organ dysfunction in the context of a second "hit"

The general identification of mutations in the structure of CFTR has been centralized for clinician reference. CFTR2.org identifies over 2000 variants of the protein, of which over 400 are disease-associated. The majority of variants are rare and not confirmed to be disease-associated, however the large number of variants indicates the lack of stability of the CFTR gene in population dynamics [1].

Classification systems of common CFTR mutations have been developed to assist with understanding of the consequential molecular defect. The established classification system includes six different classes (**Figure 2**). Different mutations can result in no functional protein production, impaired protein trafficking, altered channel gating, decreased channel conductance, reduced protein synthesis and decreased protein stability [6]. Each class confers a different disease severity, which

sustaining a functional pool of CFTR at the membrane level [15].

**40**

*CFTR classification table. The classification systems divide mutations into discrete groups determined by the predominant CFTR defect. However, these systems may not be mutually exclusive for all mutations. For example, the p.Phe508del-CFTR is predominately class II but does also have some class III and class VI properties. Reproduced with permission of the © ERS 2020: European Respiratory Journal; DOI: 10.1183/13993003.02443-2019: Accepted for publication and in press.*

is related to the degree of CFTR dysfunction and has prognostic implications for patients. However, each mutation may have features of more than just one class. For example, Phe508del is predominately a class II mutation but also has both class III and class VI properties. More recently, other classification systems have been proposed, which subdivide class I mutations (no functional CFTR protein) into two groups so as to take into account whether the mutation leads to no mRNA or no functional protein [23].
