Genetically engineered approaches to the treatment of cystic fibrosis

Abstract

Failure of functions of CFTR (cystic fibrosis transmembrane conduction regulator) gene, which encodes a protein of a selective ion channel, is causing cystic fibrosis. Cystic fibrosis is a severe systemic monogenic disease with an autosomal recessive type of inheritance, which significantly reduces the duration and quality of life of patients. It is one of the most common hereditary diseases. Studying of molecular functions of CFTR protein in different types of cells, its structural and functional network interactions are critically important for the development of a new and more effective pathogenetic therapy. We are reviewing papers on the structure of the CFTR protein and its pathogenic genetic variants, as well as methods of pathogenetic therapy of cystic fibrosis by CFTR modulators and gene engineering. Recent gene engineering approaches to keep CFTR functions are discussed, such as gene-replacement therapy and genome editing, as well as viral and non-viral delivery systems and strategies of genomic editors.

Introduction

Cystic fibrosis (CF, ICD-10 code: E84) is a severe systemic monogenic disease with an autosomal recessive type of inheritance (AR) caused by mutations in the CFTR (cystic fibrosis transmembrane conduction regulator) gene and characterized by damage to all exocrine glands as well as internal organs and systems, primarily respiratory, digestive, and reproductive (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021; Plasschaert et al. 2024). The CFTR gene consists of 27 exons and is located at locus 7q31.2, in the middle of the long arm of chromosome 7. It encodes the CFTR protein, a selective ion channel consisting of 1480 amino acids belonging to the ABCC subfamily of the ABC transporter superfamily (ATP-binding cassette transporter superfamily) (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021; Dean et al. 2022; Hanssens et al. 2021). CFTR is localized on the plasma membrane in the apical part of epithelial cells forming the lining of exocrine glands, in particular, sweat, salivary glands, bronchial glands, pancreas, intestinal glands, urogenital tract, and plays an important role in regulating the transport of Cl⁻ and HCO3⁻ ions, as well as water between cells and intercellular fluid and maintaining water balance (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021; Hanssens et al. 2021). Mutations in the CFTR gene lead to the synthesis of the defective CFTR protein, which is unable to properly carry out ion exchange or disrupt its transport to the apical membrane of epitheliocytes (Kondratyeva et al. 2023). This leads to serious disruption of epithelial cells, including mucociliary transport, increased chloride concentrations in sweat, as well as disruption of internal organs, especially the lungs, pancreas, and intestines (Lee et al. 2021). As a result, patients with cystic fibrosis accumulate excessively thick and viscous secretions in the bronchi and ducts of the exocrine glands, the microbiome of the respiratory tract is disrupted, respiratory and cardiovascular insufficiency develops, and tolerance to infections decreases (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021). The quality and life expectancy of patients are seriously decreasing (Kondratyeva et al. 2023; Frost et al. 2025). In addition, cystic fibrosis is one of the most common hereditary diseases, with a frequency from 1:600 to 1:17,000 among newborn Caucasians (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021). According to the “Register of patients with cystic fibrosis in the Russian Federation,” the incidence of cystic fibrosis in Russia is 1:9,000 newborns, the most common, with an allelic frequency of 51.4%, is the pathogenic variant CFTR—F508del (dbSNP: rs113993960) (Kondratyeva et al. 2023).

Since the first description of this disease in 1938 and the identification of the CFTR gene in 1989, significant progress has been made in understanding the etiology and pathogenesis of cystic fibrosis (Riordan et al. 1989). There is a steady increase in the number of publications on this topic (Fig. 1).

Fig. 1.

Dynamics of publication number on “cystic fibrosis gene therapy CFTR” by years by PubMed data

Figure 1 shows the increase in publications number on cystic fibrosis in the last decade (since 2015) that may relate to development of sequencing technologies and detailed studying of CTFCR gene variants.

Innovative treatment methods that directly affect the primary defects in the mutant CFTR protein are actively being developed (Terlizzi and Lopes-Pacheco 2025): note CFTR modulators (elecsacaftor-thesacaftor-ivacaftor, elexacaftor–tezacaftor–ivacaftor, ETI, etc.) (Terlizzi and Lopes-Pacheco 2025; Kondratyeva et al. 2023; Frost et al. 2025). Supportive care is also being improved. As a result, for some patients receiving treatment, it is possible to achieve a life expectancy of 72 years, which is 30 years longer than in the absence of CFTR modulators (Kondratyeva et al. 2023; Frost et al. 2025). However, despite the successes achieved and the emergence of pathogenetic therapy, the problem of cystic fibrosis remains unresolved. A high number of patients around the world continue to suffer from manifestations and complications of cystic fibrosis, as well as concomitant diseases, and the risk of premature death is still high. Therefore, it is very important to develop new and more effective methods of pathogenetic treatment, and gene therapy is a very promising approach. Since cystic fibrosis is a monogenic disease, timely correction of the mutation eliminates the cause of its development.

Etiology and pathogenesis

CFTR gene and its pathogenic variants

The CFTR gene was identified in 1989 (Riordan et al. 1989). Since then, we have managed to understand quite a lot about its structure, variants, and mechanisms of regulation of its expression. CFTR gene is located at locus 7q31.2, on the long arm of chromosome 7, and has a length of about 215 kb and contains 27 exons (Lee et al. 2021; Bareil and Bergougnoux 2020). Currently, more than 2100 mutations have been described throughout its entire length, which affect the expression of the CFTR protein to varying degrees. About 360 of these mutations are pathogenic and lead to the cystic fibrosis development (Lee et al. 2021; Bareil and Bergougnoux 2020; Jackson et al. 2023; Kiseleva et al. 2020). Active attempts are underway to create a holistic classification of CFTR gene mutations based on the defect being caused. Different groups of scientists distinguish from 5 to 7 classes of CFTR mutations (Table 1). Note that the patients can be carriers of either two identical mutations or different mutations from the same or different classes, which may complicate the selection of gene therapy as well as the choice of CFTR modulators (Plasschaert et al. 2024).

Table 1.

Classification of mutations in the CFTR gene. Adapted from Plasschaert et al. (2024); Lee et al. 2021; Kondratyeva et al. 2023; Jackson et al. 2023; Lomunova and Gershovich 2023; Kim and Krainer 2023 )

Class	Defect type	Effect	Representative variants	Carrier frequency (at least one mutation), %
I	Lack of protein synthesis	The CFTR protein is either completely absent or synthesized in a shortened form, unable to conduct ions and rapidly degrades	G542X; W1282X; R553X; CFTRdele2,3	10–22
II	Disruption of protein folding and processing	Results in the formation of an improperly folded protein that cannot be transported to the membrane	F508del; N1303K; A455E; A559T	88
III	Disruption of channel opening	The mutant CFTR does not open in response to ATP binding; the channel remains predominantly closed	G551D; S549N; G1224E; S1255P	6
IV	Reduced channel conductivity	Reduces the intensity of ion current through an open CFTR	R117H; R334W; D1152H; R347P	6
V	Lack of CFTR on the apical membrane	Functional CFTR is synthesized in insufficient quantities or is rapidly removed from the membrane	3849 + 10kbC > T (c.3718-2477C > T); A455E;	5–6
VI	Decreased stability and activity of CFTR	Mutant CFTR is unstable and poorly retained on the membrane	120del23; Q1412X; S492F	5

Table 2 shows common variants of the CFTR gene mutations in Russia according to the Registry of Patients with Cystic Fibrosis (Kondratyeva et al. 2023). F508del variant (class II) is most common (Tables 1 and 2).

Table 2.

Top 15 most common variants of the CFTR gene according to the Registry of Patients with Cystic Fibrosis in the Russian Federation (in 2023). Adapted from Kondratyeva et al. (2023)

No	Genetic variant common name	Name by coding DNA	Name by protein	Accession dbSNP	Allel frequency, %
1	F508del	c.1521_1523delCTT	p.(Phe508del)	rs113993960	51.4
2	CFTRdele2,3	c.54-5940_273 + 10250del21kb	p.(Ser18Argfs*16)	-	6.1
3	E92K	c.274G > A	p.(Glu92Lys)	rs121908751	3.71
4	1677delTA	c.1545_1546delTA	p.(Tyr515*)	rs121908776	2.51
5	3849 + 10kbC- > T	c.3718-2477C > T	-	rs75039782	2.24
6	2143delT	c.2012delT	p.(Leu671*)	rs121908812	1.99
7	2184insA	c.2052_2053insA(c.2052dupA)	p.(Gln685Thrfs*4)	rs121908786	1.93
8	W1282X	c.3846G > A	p.(Trp1282*)	rs77010898	1.73
9	L138ins	c.413_415dupTAC	p.(Leu138dup)	rs397508679	1.71
10	N1303K	c.3909C > G	p.(Asn1303Lys)	rs80034486	1.62
11	G542X	c.1624G > T	p.(Gly542*)	rs113993959	1.47
12	394delTT	c.262_263delTT	p.(Leu88Ilefs*22)	rs121908769	0.92
13	L467F;F508del#	c.[1399C > T;1521_1523delCTT]	p.[Leu467Phe;Phe508del]	-	0.87
14	R334W	c.1000C > T	p.(Arg334Trp)	rs121909011	0.75
15	W1282R	c.3844 T > C	p.(Trp1282Arg)	rs121908805	0.63

Mutations belonging to classes I, II, III, and VI are considered the most unfavorable and lead to the complete or almost complete absence of the CFTR protein and its activity causing the most severe forms of cystic fibrosis (Yeh et al. 2019). In the presence of mutations belonging to classes IV and V, the activity of CFTR is greatly reduced, but it is expressed; therefore, they are considered more “mild” variants (Kondratyeva et al. 2023).

Class I mutations

Mutations in this class lead to the complete absence of the functional CFTR protein: it is either not expressed at all or a strongly shortened variant is synthesized, which is unable to conduct ions and rapidly degrades (Fig. 2). Mutations can lead to the appearance of a premature stop codon (nonsense mutations), for example, variant G542X—glycine substitution in 542 position on the stop codon, readout frame shifts are also possible (Plasschaert et al. 2024; Lee et al. 2021; Kondratyeva et al. 2023; Jackson et al. 2023; Lomunova and Gershovich 2023). Some researchers identify the seventh class of cystic fibrosis-causing mutations that lead to the inability of CFTR mRNA formation and include the CFTRdele2.3 variant, which is common among ethnic Russians, an extended deletion affecting 21,080 base pairs: about 25% of intron 1, the entire exon 2, intron 2, and exon 3, as well as about 45% of intron 3, which causes the reading frame to shift (Plasschaert et al. 2024; Lee et al. 2021; Kondratyeva et al. 2023; Jackson et al. 2023; Kondrateva et al. 2022; Dörk et al. 2000; Park et al. 2020). However, there is evidence that with the CFTRdele2.3 variant, mRNA can still be synthesized, and even a non-functional peptide with a length of 32 amino acid residues is translated on its matrix (Kondrateva et al. 2022; Dörk et al. 2000; Park et al. 2020). Therefore, this issue requires further study. Class I mutations lead to the most severe forms of cystic fibrosis, and they occupy second place in terms of prevalence, after class II mutations.

Fig. 2.

Classification of mutations in the CFTR gene. I, class I mutations (lack of protein synthesis, G542X; W1282X; R553X; CFTRdele2,3); II, class II mutations (violation of protein folding and processing, F508del; N1303K; A455E; A559T); III, class III mutations (violation of channel opening, G551D; S549N; G1224E; S1255P); IV, class IV mutations (decreased channel conductivity, R117H; R334W; D1152H; R347P); V, class V mutations (lack of CFTR on the apical membrane, 3849 + 10kbC > T; A455E); VI, class VI mutations (decreased stability and activity of CFTR, 120del23; Q1412X)

Class II mutations

These mutations are the most common genetic variants that lead to cystic fibrosis. They lead to a violation of the folding and subsequent processing of the CFTR protein. As the result most of the synthesized CFTRS have abnormal posttranslational modifications and structure and therefore are not transported to the plasma membrane or quickly removed from it and disposed of Plasschaert et al. (2024); Lee et al. 2021; Kondratyeva et al. 2023; Jackson et al. 2023; Lomunova and Gershovich 2023) (Fig. 2). However, about 1% proteins reach the plasma membrane, but at the same time, due to the disrupted tertiary structure, they provide ion transport less efficiently (Lomunova and Gershovich 2023). Such genetic variants also lead to severe forms of cystic fibrosis. The most common mutation of this class is F508del, a phenylalanine deletion at position 508, located in the first nucleotide-binding domain (NBD1, nucleotide-binding domain 1), in the region adjacent to the first transmembrane domain (TMD1, transmembrane domain 1). The F508del mutation leads to destabilization of the CFTR structure due to disruption of the NBD1 and TMD1 domain contact. At the same time, CFTR is not transported to the membrane after translation and is utilized in proteosomes (Meng et al. 2018; Hwang et al. 2018; Grove et al. 2011). However, about 1% of CFTR can reach the plasma membrane, but their conductive function will also be reduced, and they will be removed from the membrane into the endosomes.

Class III mutations

Class III mutations lead to a violation of the opening of the CFTR channel during ATP binding, then the channel will predominantly remain closed (Plasschaert et al. 2024; Lee et al. 2021; Kondratyeva et al. 2023; Jackson et al. 2023; Lomunova and Gershovich 2023) (Fig. 2). Among the class III mutation variants, there are often missense mutations; the G551D mutation is the most common (in the NBD1 domain, glycine is replaced at position 551 by aspartic acid), which prevents the binding of ATP and the convergence of nucleotide-binding domains, which makes the channel open worse (Meng et al. 2018).

Class IV mutations

These mutations lead to a violation of the channel’s conductivity; as a result, the duration of its opening and the intensity of the ion current through the open CFTR decrease (Plasschaert et al. 2024; Lee et al. 2021; Kondratyeva et al. 2023; Jackson et al. 2023; Lomunova and Gershovich 2023) (Fig. 2). The most common variant of this class is R117H, replacing arginine at position 117 with histidine in the first extracellular loop (ICL1) resulting in no hydrogen bond forming between the arginine 117 side chain and the carbonyl group of glutamate 1124 (R117 and E1124). Patients with these class mutations usually have milder forms of cystic fibrosis.

Class V and VI mutations

A few papers have been published about the mutations of classes V and VI. It has been found that they cause milder forms of cystic fibrosis, presumably because in these cases the CFTR protein can be expressed, although in insufficient quantities to cover all needs (Duguépéroux and Braekeleer 2005). Class V mutations lead to the fact that CFTR, which can conduct ions, is synthesized in insufficient quantities or is rapidly removed from the membrane (Plasschaert et al. 2024; Lee et al. 2021; Kondratyeva et al. 2023; Jackson et al. 2023; Lomunova and Gershovich 2023) (Fig. 2). Of these, the most common mutation is 3849 + 10kbC > T (Ayupova et al. 2024; Kim and Krainer 2023). As a result of this mutation, the splicing site in intron 19 is disrupted, which leads to the insertion of an additional region (pseudoexone) with a length of 84 base pairs containing a stop codon into the mRNA, but in addition to mutant mRNAs, a small number of normal transcripts are formed (Duguépéroux and Braekeleer 2005; Kerem et al. 2021). The A455E variant is very rare and shortens the half-life of CFTR, which leads to a decrease in the amount of mature and functional CFTR on the cell surface to 12% of the norm (Berkers et al. 2021). Class VI mutations are very rare; they reduce the stability of the CFTR protein, which is why it is poorly retained on the membrane (Fig. 2). Variant 120del23—deletion of 23 nucleotides containing the start codon (positions 133–135) leads to the formation of alternative codons “AUG” (M150 or M152 or M156), truncated from the N-terminus of the CFTR protein (Ramalho et al. 2009). At the same time, the 120del23 mutation does not affect the level of transcription, and mRNA from the CFTR gene is synthesized in normal amounts, but the protein translated from them is unstable, exhibits a very low ability to conduct Cl⁻, is poorly retained on the membrane, and has abnormal post-translational modifications due to a defect in the N-terminus necessary for proper maturation (Ramalho et al. 2009). As a result of the Q1412X mutation, a CFTR variant is expressed that lacks the last two β-chains in the NBD2 domain and the entire C-terminal region, which is very poorly retained on the membrane, but, in addition, due to the dysfunction of the ATP-binding site in NBD2, has a 95% reduced probability of opening (Yeh et al. 2019).

Currently, the diagnosis of cystic fibrosis is based on a positive result of neonatal screening (NBS), the presence of clinical signs characteristic of this disease, or a positive family history in the presence of a high level of SCC and/or two pathogenic variants of the CFTR gene (Terlizzi and Lopes-Pacheco 2025).

CFTR protein, its functions, and role in pathogenesis

The CFTR protein is a transmembrane glycoprotein consisting of 1480 amino acid residues and belonging to the ABCC subfamily of the ABC transporter superfamily (Dean et al. 2022; Hanssens et al. 2021). CFTR is localized on the plasma membrane in the apical part of epithelial cells forming the lining of exocrine glands, in particular, sweat, salivary glands, bronchial glands, pancreas, intestinal glands, and urogenital tract. This protein performs the function of a selective ion channel and plays an important role in regulating the transport of Cl⁻ and HCO3⁻ ions, as well as water between cells and the intercellular fluid and, as a result, in regulating the processes of absorption and secretion (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021; Hanssens et al. 2021; Lomunova and Gershovich 2023). In response to ATP binding, CFTR changes its conformation, and the channel opens, which allows the Cl⁻ ion to passively move through the membrane, then after ATP hydrolysis, the channel closes (Hanssens et al. 2021; Lomunova and Gershovich 2023; Simon and Csanády 2021; Levring et al. 2023; Lei et al. 2023). CFTR is the only ABC family channel providing passive ion transport (McDonald et al. 2023). CFTR has a domain structure and contains two nucleotide-binding (NBD1 and NBD2) and two transmembrane (TMD1 and TMD2) domains; NBD1 is directly connected to TMD1, which is located at the N-terminus and begins with a lasso motif containing two α-helices half-immersed in the plasma membrane from the cytoplasm and an α-helix wrapped around TMD2, which is connected to NBD2, located at the C-end. (McDonald et al. 2023). Each of the transmembrane domains consists of six hydrophobic transmembrane α-helices (TM) (Fig. 3). The TM1–TM6 helices are located in transmembrane domain 1 (TMD1), and TM7–TM12 are in TMD2. Unlike other ABC family proteins, CFTR has a cytoplasmic unstructured regulatory domain (R domain) between NBD1 and TMD2 (Simon and Csanády 2021; McDonald et al. 2023). Transmembrane domains form the ion channel of the CFTR protein, and NBD provides its opening and closing in response to ATP binding and hydrolysis; the R domain separates the TMD domains (Hanssens et al. 2021; Simon and Csanády 2021; Levring et al. 2023; McDonald et al. 2023). If the R domain is in a dephosphorylated state and in the absence of ATP, the CFTR forms a closed conformation in which the NBDs are approximately 20 Å apart, and the R domain sterically prevents their convergence (Levring et al. 2023). The opening of the CFTR channel occurs in response to phosphorylation of the R domain by protein kinase A (PKA, PKA) and binding of ATP to NBD, which leads to dimerization of the NBD1 and NBD2 domains, a change in the conformation of the TMD domains from the inward to the outward conformation, and the release of Cl ions from epithelial cells. After ATP hydrolysis, the NBD dimer diverges, the TMD conformation is restored, and the CFTR channel closes (Simon and Csanády 2021; Levring et al. 2023).

Fig. 3.

The structure of dephosphorylated, non-ATP-bound CFTR. Adapted from McDonald et al. (2023)

The presence of Cl⁻, HCO₃⁻, and Na⁺ ions in the intercellular space near the apical surface of epithelial cells is a necessary condition for maintaining normal osmotic pressure and is necessary for fluid circulation near the plasma membranes of cells (Lomunova and Gershovich 2023; Lei et al. 2023). It is noteworthy that CFTR is also involved in the turnover of the HCO3⁻ ion, which plays an important role in maintaining homeostasis: intracellular and extracellular pH of the respiratory tract (Hanssens et al. 2021; Lomunova and Gershovich 2023; Tümmler 2023). In addition, for the normal functioning of cells, a constant and controlled flow of these ions through the membrane is required, which is provided by membrane transporters, which include CFTR. This channel regulates ion transport in the ciliated epithelium of the respiratory tract and submucosal glands: as a result, the amount of fluid on the surface of the respiratory tract and the degree of viscosity of mucus (Tümmler 2023).

A decrease in the concentration of Cl⁻ ions near the surface of epitheliocytes, which occurs when the CFTR is disrupted, activates the epithelial sodium channel (ENaC), which pumps Na⁺ ions into cells. The lack of Na⁺ and Cl⁻ ions in the intercellular fluid near epitheliocytes leads to a decrease in the volume of water entering the cell, which in turn leads to the formation of a thicker and more viscous secretion, and loss of the ability of the respiratory tract to self-purify due to disruption of the cilia of the cells of the mucous membrane. Mucus stagnates in the lumen of the glands, which disrupts their work and reduces the body’s resistance to infections (Lomunova and Gershovich 2023).

Ionocytes

Ionocytes are cells with the highest level of CFTR expression. We highlight their role in regulating fluid secretion and adsorption in the respiratory tract. CFTR protein is expressed by only a few percent of the cells of the respiratory tract epithelium in human. Most of such cells are secretory cells (Tümmler 2023). In 2018, according to single-cell RNA sequencing, a small population of cells called ionocytes was identified, accounting for only 0.3–1% of the total number of epitheliocytes (Plasschaert et al. 2018). They are actively involved in the regulation of ASL secretion and are characterized by the highest level of CFTR expression: about half of all mRNA transcripts of the CFTR gene originate from ionocytes (Li et al. 2019; Tümmler 2023; Montoro et al. 2018; Plasschaert et al. 2018; Thornell et al. 2025). Secretory cells contain about 10 times fewer CFTR gene transcripts than ionocytes, but they make up more than 10% of the total number of respiratory tract epithelial cells (Lei et al. 2023).

Therefore, immediately after the discovery, ionocytes attracted the close attention of the scientific community. Active studies of their functions and mechanisms of ion transport mediated by them have begun. Understanding this will allow us to form a complete picture of the pathogenesis of cystic fibrosis and find new targets for more effective therapy. However, then contradictory publications began to appear on the direction of Cl^– ion transport through CFTR and the role of these cells in regulating ASL levels. In studies conducted using the short-circuit current technique, it was found that, at a transepithelial voltage of 0 mV, CFTR channels in ionocytes mediate the secretion of Cl⁻. In studies that measured the absorption of liquid or the gradient of Cl⁻ ions on the apical surface of ionocytes, that is, in more physiological open-circuit conditions, it was noted that CFTR channels are responsible for the absorption of Cl⁻ ions (Montoro et al. 2018).

The key difference between these experiments is the presence or absence of a transepithelial electrochemical gradient. Without it, as with the short-circuit current method, only active ion transport through the epithelium is possible (Montoro et al. 2018). In open-circuit conditions, the transepithelial electrochemical gradient takes on negative values, which leads to the absorption of Cl⁻ ions through CFTR channels (Lei et al. 2023; Montoro et al. 2018).

In addition, it has recently been found that an increase in the number of ionocytes achieved by transformation of the culture of epithelial cells of the respiratory tract by lentivirus, which overexpresses the transcription factor FOXI1 (forkhead box I1), increases fluid uptake, and their decrease caused by disruption of the FOXI1 gene increases secretion and reduces fluid uptake (Lei et al. 2023; Tümmler 2023; Montoro et al. 2018; Thornell et al. 2025). At the same time, a decrease in the amount of CFTR on the ionocyte surface leads to a decrease in fluid absorption (Romano Ibarra et al. 2024). These data contradict the previously proposed hypothesis that the main function of ionocytes is the secretion of Cl⁻ ions, and, as a result, an increase in ASL volume (Montoro et al. 2018).

Thus, it was concluded that the direction of Cl⁻ ion transfer through CFTR is determined by the electrochemical gradient and the concentration gradient of Cl⁻. Ionocytes can both absorb and secrete Cl⁻, depending on the conditions in which they are located, as well as on the activity of other ion channels on their plasma membrane. In the intestinal epithelium and sweat gland ducts, the transport of Cl⁻ ions through CFTR proceeds in two directions (Montoro et al. 2018).

In addition to CFTR, many different ion channels function on the ionocyte membrane. The basolateral membrane contains Na⁺/K⁺-ATPases (Na–K pumps) and specific barttin/Cl^– channels (ClC–K/barttin channels) for this type of cell. Barttin is a regulatory subunit of ClC-type chloride channels characteristic of humans. Barttin is encoded by the BSND gene; mutations in this gene lead to Bartter’s syndrome types IV, the clinical manifestations of which include congenital deafness and nephropathy associated with salt loss, but do not lead to pulmonary manifestations characteristic of cystic fibrosis (Tümmler 2023; Mrad et al. 2021). There are ENaC (epithelial sodium channel) and CFTR on the apical membrane (Lei et al. 2023; Tümmler 2023; Montoro et al. 2018). It is ENaC and Na⁺/K⁺-ATPases that create a transepithelial electrochemical gradient, which promotes the absorption of ions by Cl^– ionocytes, and, as a result, the absorption of liquid from ASL, which leads to a decrease in its volume (Tümmler 2023).

It is noteworthy that disruption of the barttin/Cl^– channels and removal of barttin reduces fluid absorption and increases ASL volume, while overexpression increases fluid absorption and decreases ASL volume (Lei et al. 2023). Secretory cells, on the contrary, absorb Cl^– ions through basolateral NKCC1 channels and secrete them through apical CFTR. The coordinated work of the apical CFTR channels and basal barttin/Cl^– channels ensures the normal exchange of Cl^– ions through ionocytes, and the transepithelial tension created by ENaC stimulates fluid absorption.

Thus, CFTR perform opposite functions in different cell types: in ionocytes, they ensure the import of Cl^– ions and play a key role in the absorption of fluid from the apical part of the ASL and in secretory cells—in its secretion (Lei et al. 2023; Tümmler 2023) (Fig. 4). In cystic fibrosis, not only secretion is impaired but also absorption of fluid in the respiratory tract (Lei et al. 2023). The appearance of data on the distribution of CFTR between different types of respiratory tract epitheliocytes and the clarification of their role in the regulation of Cl^– ion transport and ASL volume raises the question of whether gene therapy for cystic fibrosis should target a specific type of cell, and if so, which one. It has been hypothesized that the most promising strategy for gene therapy of cystic fibrosis is to eliminate mutations in the CFTR gene specifically in secretory cells. And if stem cells, which, for example, include basal cells, are selected as a target, it is possible to restore CFTR functions in both secretory cells and ionocytes (Plasschaert et al. 2024; Lei et al. 2023; Tümmler 2023).

Fig. 4.

The coordinated operation of ion channels determines the normal level of ASL and mucociliary transport. Ionocytes and secretory cells perform opposite functions and provide a flow of Cl⁻ ions in opposite directions: A normal; B for cystic fibrosis

The secret of the epithelial cells of the respiratory tract—pulmonary surfactant (ASL, airway surface liquid)—is mucus, consisting mainly of O-glycosylated glycoproteins, mainly mucins, forming a protein network on the surface of the epithelium. ASL also contains antimicrobial peptides, which are part of the innate immune system and are involved in protecting the lungs from bacteria (Romano Ibarra et al. 2024). The normal porosity and viscosity of ASL ensure the capture and removal of inhaled particles and bacteria from the respiratory tract, as well as the free migration of neutrophils. Therefore, in cystic fibrosis, bacteria are able to form stable colonies on the surface of the epithelium. The secreted lower respiratory tract contains S. aureus, P. aeruginosa, B. cepacia complex, S. maltophilia, Achromobacter spp., and respiratory pathogens such as H. influenzae, S. pneumoniae, M. Catarrhalis, and many other microorganisms, including fungi: representatives of the genus Aspergillus (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021).

In cystic fibrosis, impaired ion transport through the CFTR leads to a decrease in porosity and an increase in the viscosity of ASL, which leads to disruption of the function of epithelial cells of the lungs and upper respiratory tract, as well as the pancreas, liver, gallbladder, intestines, sweat glands, and reproductive tract (Hanssens et al. 2021; Lomunova and Gershovich 2023). Therefore, cystic fibrosis is a multisystem disease with diverse and severe manifestations (Table 3).

Table 3.

Clinical manifestations of cystic fibrosis. Adapted from Hanssens et al. (2021)

Organs	Manifestations of cystic fibrosis
Lungs	Lung airway obstruction, chronic bacterial infection, bronchiectasis, pneumothorax, hemoptysis
Nasal sinuses	Sinusitis, polyps
Intestine	Meconium intestinal obstruction, distal intestinal obstruction syndrome, rectal prolapse
Liver	Obstructive diseases of the biliary tract
Pancreas	Exocrine insufficiency, diabetes associated with cystic fibrosis
Sweat glands	Increased levels of chloride in sweat
Reproductive system	Male and female infertility

Pathogenic therapy

To date, significant progress has been made in understanding the pathogenesis and development of therapy against cystic fibrosis, and the survival rate and quality of life of patients are significantly increasing. With the right therapy, the average life expectancy for people with this disease is 40 or even more than 50 years (Tümmler 2023). The FDA has already approved a strategy for the treatment of cystic fibrosis based on the use of small molecules—CFTR modulators, capable of restoring its function to a certain extent in the presence of the most common class II–VI mutations found in 90% of patients. However, 10% of the variants causing cystic fibrosis are resistant to the action of CFTR modulators because in such cases either CFTR protein synthesis is completely absent or it is too defective to provide ion transport (Plasschaert et al. 2024). Moreover, 10–20% of patients have individual intolerance to CFTR modulators (Maule et al. 2020; Lomunova and Gershovich 2023). According to the mechanism of action, CFTR modulators are divided into potentiators, correctors, stabilizers, amplifiers, and read-through agents (through agents, substances that promote the “reading” of premature stop codons) (Lee et al. 2021; Terlizzi and Lopes-Pacheco 2025; Maule et al. 2020; Lopes-Pacheco 2020). Currently, four CFTR modulators are clinically approved: the ivacaftor potentiator (VX-770) as monotherapy or as part of combination therapy with correctors, lumacaftor (VX-809), tezacaftor (VX-661), and elecsacaftor (VX-445) (Baroni 2025; McDonald et al. 2023).

Potentiators

Potentiators include compounds that increase the probability of opening a CFTR channel, thereby increasing the CFTR-mediated ion flow through the plasma membrane (Lopes-Pacheco 2020). Potentiators are effective against class III and IV mutations (R117H, R334W, R347P, and G551D), which occur infrequently and disrupt CFTR function, but do not reduce its expression level (Lopes-Pacheco 2020). These compounds bind to CFTR at certain sites, contributing to the proper opening of the channel and increasing the activity of CFTR already present on the membrane. Examples of potentiators are ivacaftor and dirocaftor (PTI-808) (Lee et al. 2021; Terlizzi and Lopes-Pacheco 2025; Maule et al. 2020).

Ivacaftor (Kalydeco™, VX-770, PubChem CID: 16220172), manufactured by the German company Vertex Pharmaceuticals, is the first pathogenetic drug against cystic fibrosis (Fig. 5), which was approved by the FDA and EMA in 2012 (Terlizzi and Lopes-Pacheco 2025; Goor et al. 2009; Eckford et al. 2012).

Fig. 5.

The structural formula of ivacaftor. Ivacaftor is an aromatic amide obtained by condensation of the carboxyl group of 4-oxo-1,4-dihydroquinoline-3-carboxylic acid with the amino group of 5-amino-2,4-di-tert-butylphenol. It contains quinolone and phenol residues, as well as an amide group that ensures the formation of hydrogen bonds, which determines activity, pharmacokinetics, and pharmacodynamics (Ferreira et al. 2024)

Ivacaftor ensures the correct opening of the CFTR channel by stabilizing the open conformation, thereby increasing the secretion of Cl⁻ and bicarbonate ions (Goor et al. 2009; Eckford et al. 2012; Ferreira et al. 2024). This drug has proven to be safe and effective. It is noteworthy that in the 2010 s, this drug was used to treat patients over 2 years of age with the G551D mutation, whereas since 2017, the FDA has approved its use for a wider range of variants, including 38 mutations: E56K, G178R, S549R, K1060T, G1244E, 3272-26A- > G, P67L, E193K, G551D, A1067T, S1251N, 711 + 3A- > G, R74W, L206W, G551S, G1069R, S1255P, E831X, D110E, R347H, D579G, R1070Q, D1270N, D110H, R352Q, S945L, R1070W, G1349D, R117C, A455E, S977F, F1074L, 3849 + 10kbC- > T, R117H, S549N, F1052V, D1152H, 2789 + 5G- > A (Baroni 2025; FDA approves ivacaftor for five splice mutations 2017).

The exact mechanism of action of ivacaftor remained unknown for a long time. In 2019, cryo-electron microscopy was used to determine the structure of the CFTR complex with ivacaftor (PDB: 6O2P), which made it possible to establish two binding sites for this drug (Fig. 6). Ivacaftor binds to CFTR inside the lipid bilayer between TMD1 and TMD2, the amino acid residues Phe931 and Arg933 play a key role in this, as well as in the region of the intracellular loop ICL4 (intercellular loop 4) at the boundary of NBD1 and TMD2 (Ferreira et al. 2024; Baroni 2025; McDonald et al. 2023).

Fig. 6.

The structure of phosphorylated, ATP-bound human CFTR protein in complex with ivacaftor (Ferreira et al. 2024)

However, the use of ivacaftor alone for the treatment of patients with the F508del mutation in the homozygous state proved to be insufficiently effective due to a lack of CFTR on the cell membrane. In such cases, combination therapy is necessary, including a CFTR corrector (Connett 2019).

Correctors

Compounds that improve CFTR folding and, therefore, facilitate processing and transport to the apical membrane of cells are referred to as correctors (Lee et al. 2021; Terlizzi and Lopes-Pacheco 2025; Maule et al. 2020; Lopes-Pacheco 2020; Kim et al. 2023). Disruption of these processes is observed in the presence of the most common mutations that cause cystic fibrosis, which belong to class II (F508del, G85E, I507del, R560T, and N1303K). Most correctors, acting by various mechanisms, increase the conformational stability of mutant CFTR during folding into EPR, acting as pharmacological chaperones (Lopes-Pacheco 2020). Correctors at the site of binding to CFTR are divided into three types (Baroni 2025; Kim et al. 2023; McDonald et al. 2023) (Fig. 7). Type I correctors, such as lumacaftor (VX-809) and tezacaftor (VX-661), stabilize the CFTR site between NBD1 and TMD1; type III correctors, which include elexacaftor (VX-445), bind to the transmembrane helices TM2, TM10, TM11, and the lasso motif, allosterically stabilizing NBD1 (Baroni 2025; Kim et al. 2023; McDonald et al. 2023). The binding sites of type II correctors are not yet known, but it has been established that they bind to NBD2 and stabilize it; at the moment none of them has yet been approved by the FDA (McDonald et al. 2023; Marchesin et al. 2024). In addition to these three types, compounds with a new CFTR folding correction mechanism were discovered in 2024, which can be isolated into type IV CFTR correctors (Marchesin et al. 2024). Most likely, type IV correctors bind to CFTR in the cavity between the lasso-1 (Lh1) and TM1 helices, which promotes the co-translational assembly of Lh1, TMD1, and TMD2 (Marchesin et al. 2024).

Fig. 7.

A Chemical structures of FDA-approved CFTR modulators. B Cryoelectronic structure of F508del CFTR associated with VX-661, VX-445, and VX-77036 (PDBID 8EIQ). C Close-up of the binding site of VX-661 in TMD1 interacting with TM1, TM2, TM3, and TM6. A salt bridge with R74 is formed at the very beginning of TM1 next to the lasso motif. D VX-445 binds to TMD2/lasso motif and interacts with TM10 and TM11, as well as with N-terminal residues in the lasso motif and TM2 (McDonald et al. 2023)

Lumacaftor (VX-809, PubChem ID: 16,678,941) is the first and one of the most common correctors, as a component of the lumacaftor/ivacaftor combination therapy (Orkambi™), approved by the FDA in 2015 for the treatment of patients over 12 years of age with the homozygous F508del mutation (Lopes-Pacheco 2020). As in the case of ivacaftor, lumacaftor is not used in monotherapy due to its low efficacy (Lopes-Pacheco 2020).

The mechanism of action of lumacaftor is also not fully understood; it may have several binding sites: in TMD1 and at the NBD1-TMD2 interface (Baroni 2025; Lopes-Pacheco 2020). To date, in Russia, the combined drug ivacaftor + lumacaftor (Orkambi™) is used to treat cystic fibrosis in patients aged 2 years and older with the F508del mutation in the homozygous state, since the effectiveness and safety of this drug have been proven only for the homozygous F508del mutation (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021).

Tezacaftor (VX-661; PubChem ID: 46,199,646) is a second-generation corrector developed by Vertex Pharmaceuticals based on lumacaftor, which shows better pharmacokinetic properties and fewer side effects (Lopes-Pacheco 2020), and is approved by the FDA for the treatment of patients over 12 years of age in combination with ivacaftor (Symdeko™). It is also part of two drugs for triple combination therapy of cystic fibrosis, elecsacaftor + tezacaftor + ivacaftor (ETI, available under the trade name Trikafta™ in the USA and Kaftrio™ in Europe), and vanzacaftor + tezacaftor + deutivacaftor (Alyftrek™) (Baroni 2025; Bengtson et al. 2022).

Stabilizers

These include compounds that restore the stability of CFTR on the plasma membrane, and prevent its endocytosis and degradation in lysosomes. (Lee et al. 2021; Terlizzi and Lopes-Pacheco 2025; Maule et al. 2020; Lopes-Pacheco 2020). Stabilizers are potentially directed against class VI mutations (Lopes-Pacheco 2020). Cavosonstat from Nivalis is the first stabilizer to reach clinical trials. It was found to be safe and reduced chloride concentrations in sweat in patients with the F508del mutation in the homozygous state, but its effectiveness was found insufficient and clinical trials were discontinued (Lopes-Pacheco 2020). The action of the stabilizers is based on various mechanisms. Cavosonstat is an inhibitor of S-nitrosoglutathione reductase (GSNOR), which catalyzes the irreversible conversion of S-nitrosoglutathione (GSNO) to oxidized glutathione (Donaldson et al. 2017). A decrease in GSNO concentration negatively affects CFTR biogenesis. GSNO modulates the function of Hsp70/Hsp90 organizing proteins (HOP) through S-nitrosation of cysteine residues. HOP participates in the regulation of CFTR biogenesis and transport (Donaldson et al. 2017).

Amplifiers

The amplifiers increase the expression of CFTR mRNA, resulting in an increase in the amount of CFTR protein, which is currently undergoing clinical trials (Lee et al. 2021; Terlizzi and Lopes-Pacheco 2025; Maule et al. 2020; Lopes-Pacheco 2020; Bengtson et al. 2022). These drugs are directed against class V mutations (3849 + 10kbC > T, 2789 + 5G > A and A455E), in which the number of CFTR mRNAs decreases. Nesolicaftor (PTI-428) from Proteostasis Therapeutics is the first amplifier capable of increasing the amount of CFTR regardless of genotype, and the best effect is achieved with combination therapy (Lopes-Pacheco 2020; Bengtson et al. 2022). Nesolicaftor directly interacts with poly(rC)-binding protein 1 (PCBP1), binding of which to CFTR mRNA increases translation efficiency (Bengtson et al. 2022; Dukovski et al. 2020; Tutone et al. 2019).

Read-through agents (through agents, substances that promote the “reading” of premature stop codons)

Drugs are actively being developed for the pathogenetic therapy of cystic fibrosis caused by mutations leading to the appearance of a premature stop codon (PTC), nonsense mutations, for example, W1282X and G542X. Read-through agents allow the ribosome to pass through the PTC by inserting an amino acid in its place, which ensures the continuation of translation and synthesis of functional CFTR (Lopes-Pacheco 2020; Lentini et al. 2014; Karri et al. 2024).

For the first time, the ability to mask PTC was found in aminoglycoside antibiotics such as gentamicin and genetycin. However, aminoglycosides are not suitable for pathogenetic therapy of cystic fibrosis because they are insufficiently effective and exhibit nephrotoxicity and ototoxicity with prolonged use (Lopes-Pacheco 2020; Lentini et al. 2014; Karri et al. 2024). Currently, compounds unrelated to aminoglycosides are being developed: ataluren (PTC124), NV2445, negamycin, clitocine, escin, 2,6-diaminopurine, and 2-guanidino-quinazoline (TLN468) (Karri et al. 2024; Tutone et al. 2019; Bidou et al. 2022). The mechanisms of action of these compounds are not fully known; it is assumed that they may have binding sites on ribosomes, such as the negamycin dipeptide, which binds to the A site, interact with rRNA or mRNA, and bind to proteins directly or indirectly involved in the translation process, such as 2,6-diaminopurine, which inhibits Ftsj1, which leads to specific suppression of premature UGA (Bidou et al. 2022).

One of the main disadvantages of read-through agents is the dependence of their effectiveness on the nature of the stop codon (UAG, UGA or UAA) and its environment; in addition, the amino acid that is located in this place in the wild-type CFTR is not always embedded in the place of PTC, so there is a risk protein formation with strongly reduced activity (Karri et al. 2024). For example, TLN468 in 95% of cases initiates the insertion of cysteine in the place of UGA (S1196X mutation) and UGA (G542X), glutamine in the place of UAG (W846X) (97%), and UAA (E1417X) tyrosine (85%) or glutamine (11%) (Karri et al. 2024).

Ataluren (Translarna™; PTC124; PubChem ID: 11,219,835) is one of the first drugs that promote the “reading” of PTC that have reached clinical trials. Its most likely target is directly premature stop codons on mRNA, and there is evidence that it can bind to rRNA (Tutone et al. 2019). Ataluren was recognized as safe and was generally well tolerated by patients. However, according to the results of phase III of clinical trials (NCT02139306), its development for the treatment of cystic fibrosis was discontinued due to low efficacy. Nevertheless, this drug has been approved for the treatment of patients with Duchenne muscular dystrophy (Lopes-Pacheco 2020).

Undoubtedly, it is possible to effectively combat the manifestations of cystic fibrosis with a combination of pathogenetic and symptomatic therapy, including drugs of various groups, including corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, and antifungal agents. Also, all patients with cystic fibrosis need diet therapy, vitamin therapy, and kinesiotherapy aimed at cleansing the bronchi of viscous sputum and including postural drainage, breathing exercises, work with special simulators, physiotherapy, and physical therapy (The Union of Pediatricians of Russia, Association of Medical Geneticists, the Russian Respiratory Society, the Russian Transplant Society, the Association of Pediatricians of the Moscow region 2021). All this significantly affects the quality of life of patients who have to take expensive medications and adjust their lifestyle daily. Treatment requires discipline, a serious and attentive approach. That is why it is so important to develop effective gene therapy that can permanently eliminate the cause and manifestations of cystic fibrosis, which will improve the lives of thousands of patients, as well as reduce the burden on the healthcare system.

Genetic engineering approaches

In contrast to CFTR modulators, which only compensate for the effect of certain mutations, and there are mutations that are not amenable to the action of modulators, gene therapy is aimed at correcting any variants that lead to the development of cystic fibrosis and is able to ensure full recovery (Maule et al. 2020).

The most severe manifestations of cystic fibrosis are associated with impaired lung function; however, the lungs are available for the delivery of gene therapy drugs through inhalation or bronchoscopy (Plasschaert et al. 2024). Difficulties in the development and application of gene therapy are mainly related to the presence of a mucus layer on the surface of the respiratory epithelium, immune reactions to delivery systems. Moreover, the epithelium of the respiratory tract is constantly being updated, which prevents the preservation of edited cells. As well as CFTR-mediated secretion and absorption of Cl^‒ ions in the lungs, they are distributed among different cell types, so there is also a problem in finding specific target cells for gene therapy of cystic fibrosis (Plasschaert et al. 2024; Lei et al. 2023; Tümmler 2023). In addition, there is currently a problem in selecting in vivo models for testing gene therapy drugs against cystic fibrosis, since it is difficult to reproduce the pathogenesis of this disease in mice with differently positioned ionocytes. Therefore, despite significant progress in genetic technologies, it has not yet been possible to create a gene therapy for cystic fibrosis, but research is actively underway in this direction and such drugs are expected to appear in the future (Table 4).

Table 4.

Clinical trials of gene therapy for cystic fibrosis

Clinical trial ID, start year	Title	Vector	Results
NCT00004779 1993	Phase I pilot study of Ad5-CB-CFTR, an adenovirus vector containing the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis	Adenovirus vector (Ad) based on recombinant adenovirus of the 5th serotype with the deleted E1 gene containing the CFTR gene	Vector expression was detected only in patients who received high doses of the drug; 3 weeks after the intervention, no mRNA was detected. The vector elicited an immune response on the nasal mucosa, < 1% of epithelial cells were transformed, and no significant restoration of CFTR functions was detected ( Knowles et al. 1995 )
NCT00004806 1995	Phase I study of liposome mediated gene transfer in patients with cystic fibrosis	Liposomes	No published results
NCT00004471 1995	Phase I pilot study of gene therapy for cystic fibrosis using cationic liposome mediated gene transfer	Cationic liposomes	No side effects; histological and immunohistological changes were detected. Partial restoration of CFTR function was achieved, but 7 days after the intervention, the effect disappeared. Plasmid DNA and RNA obtained from the vector were found in most of the patients treated
NCT00004287 Zuckerman et al. 1999) 1995	Phase I study of the third generation adenovirus H5.001CBCFTR in patients with cystic fibrosis	H5.001CBCFTR is a third-generation adenoviral vector (Ad) containing the recombinant CFTR gene	Adult volunteers with cystic fibrosis received gene therapy based on H5.001CBCFTR; the drug was injected into the respiratory tract using a bronchoscope. The efficiency of transferring the CFTR gene into bronchial epithelial cells was < 1% 4 days after instillation of the vector, by day 43 the vector was not detected, and the majority of volunteers showed a pronounced immune response
NCT00004533 Flotte et al. 2003) 1999	Phase I randomized study of adeno associated virus CFTR vector in patients with cystic fibrosis	Recombinant vector based on the adeno-associated virus of the second serotype (rAAV2-CFTR) containing the full-length CFTR gene	Volunteers over the age of 15 with cystic fibrosis received gene therapy based on rAAV2-CFTR containing the CFTR gene. Adverse events, probably unrelated to the administration of the drug, were noted; however, serious adverse reactions to the vector were noted in one patient. Anti-AAV2 antibodies were detected in some volunteers. The efficiency of CFTR gene transfer was 0.002–0.5 copies per cell
NCT00073463 2003	Safety and efficacy of recombinant adeno-associated virus containing the CFTR gene in the treatment of cystic fibrosis	A recombinant vector based on an adeno-associated virus (tgAAVCF) containing cDNA of the CFTR	tgAAVCF was administered in aerosol form to volunteers over 12 years of age with cystic fibrosis, undesirable reactions, including serious ones, were detected, the maximum efficiency of CFTR gene delivery on day 14 after administration was 0.6 copies per cell, on day 30, 0.1, and on day 90 the vector was almost undetectable ( Aitken et al. 2001 ). Sputum-induced IL-8 improved, anti-AAV2 antibodies were detected, and CFTR gene transfer was detected, but not expression ( Moss et al. 2004 ). tgAAVCF was generally well tolerated and considered safe, but did not significantly improve lung function ( Moss et al. 2007 )
NCT00789867 2008	Single dose of pGM169/GL67A in CF patients	pGM169/GL67A, a plasmid containing the CFTR gene, in complex with a mixture of cationic lipids (GL67A) consisting of a cholesterol derivative GL67, neutral lipid DOPE and pegylated lipid DMPE-PEG5000	Volunteers aged 16 to 70 with cystic fibrosis received the drug in the form of an aerosol using a nebulizer. The results have not been published
NCT01621867 2012	Repeated application of gene therapy in CF patients	pGM169/GL67A, A plasmid containing the CFTR gene, in complex with a mixture of cationic lipids (GL67A) consisting of a cholesterol derivative GL67, neutral lipid DOPE and pegylated lipid DMPE-PEG5000	Volunteers over 12 years of age with cystic fibrosis received the drug in the form of an aerosol using a nebulizer with an interval of 28 (± 5) days for 1 year. No serious adverse events were detected. There was a moderate improvement in lung function (an increase in forced exhalation volume in 1 s. and accelerated lung capacity), but not the quality of life ( Alton et al. 2016 )
NCT02564354 2015	Exploratory study to evaluate QR010 in subjects with cystic fibrosis ΔF508 CFTR mutation	Antisense oligonucleotide (QR-010, now called Eluforsen), targeting the f508del mutation	Volunteers over the age of 18 with homozygous or heterozygous F508del mutation received QR-010 internasally for 4 weeks. In the cohort with the homozygous mutation, an improvement in total chloride transport was observed, but no improvement was recorded with the heterozygous mutation. The drug was recognized as safe, although undesirable effects were detected ( Sermet-Gaudelus et al. 2019 )
NCT02532764 2015	Dose escalation study of QR010 in homozygous ΔF508 cystic fibrosis patients	Antisense oligonucleotide (QR-010, now called Eluforsen), targeting the f508del mutation	Volunteers aged 18–60 years, homozygous for the F508del mutation, divided into groups with a single dose increase (SAD) and multiple dose increases (MAD), received inhaled eluforsen using a nebulizer for 4 weeks. No dose-dependent toxic effects were detected, and, therefore, the maximum tolerated dose was not established, but undesirable effects were observed. Nevertheless, the drug was found to be safe. There was an improvement in the quality of life according to CFQ-R RSS. However, the effectiveness of the drug was insufficient, and there was no significant improvement in lung function and chloride content in sweat compared with placebo ( Kim and Krainer 2023; Drevinek et al. 2020 )
NCT05248230 2022	4D-710 in adult patients with cystic fibrosis	4D-710, an adeno-associated viral vector (AAV) consisting of a variant of the AAV capsid (4D-A101) carrying a transgenic cassette encoding CFTR	At the moment, clinical trials are ongoing, and the final results have not been published
NCT06526923 2024	A phase 1/2 trial of SP-101 for the treatment of cystic fibrosis (CF)	SP-101, a recombinant vector based on adeno-associated virus (AAV), a hybrid of AAV2 and AAV5 containing the CFTR minigene (hCFTRΔR)
NCT06515002 2024	A study to test how well BI 3720931 is tolerated and whether it improves lung function in people with cystic fibrosis (Lenticlair™)	A third-generation lentiviral vector pseudotyped with the shell proteins F and HN of the Sendai virus (rSIV.F/HN), carrying codon-optimized and CpG-sequence-free cDNA CFTR (soCFTR2) under the control of a hybrid promoter without CpG sequences (hCEF) (rSIV.F/HN-hCEF-soCFTR2)
NCT06962852 2025	A long-term study to monitor the health status of people with cystic fibrosis who took part in a previous study with BI 3720931 (Lenticlair™-ON)

Gene-replacement therapy

Gene-replacement therapy is a field of gene therapy that involves transferring a functional copy of a gene or mRNA encoding the functional product of this gene into cells in order to synthesize its product. The delivery of nucleic acids to certain cells is not an easy task and is solved by using various delivery systems or vectors. Vectors can be divided into viral and non-viral ones. Usually, viral vectors able to deliver genetic material into cells more efficiently than non-viral ones.

Viral delivery systems

The very first attempts to create a gene therapy against cystic fibrosis in 1993–2001 (NCT00004779, NCT00004287) were made using vectors based on adenovirus (Ad) (Plasschaert et al. 2024; Lee et al. 2021). To create a vector, replication and immune evasion genes were removed from the adenovirus, which deprived it of the ability to reproduce itself (Lee et al. 2021; Maeder et al. 2008). The main advantage of adenoviruses as vectors is that they do not integrate into the genome, and therefore, when used, there is less risk of oncogenesis induction and inappropriate editing (Maeder et al. 2008). However, adenoviral vectors elicited a strong immune response and delivered genetic material to epithelial cells with low efficiency, since they cannot penetrate basal cells, and therefore did not provide a long-term effect of gene therapy (Plasschaert et al. 2024; Lee et al. 2021). To increase the efficiency of transduction and reduce immune responses to the vector, other studies used helper-dependent adenoviruses (Hd-Ad) in which all viral genes have been removed (Lee et al. 2021; Lomunova and Gershovich 2023; Davies et al. 2024; Xia et al. 2019). At the same time, Hd-Ad was delivered to the lungs along with lysophosphatidylcholine (LPC) for better passage of the mucus layer, which allowed for increased editing efficiency and increased expression time of the delivered copy of the CFTR gene (Lee et al. 2021; Lomunova and Gershovich 2023). However, currently adenoviruses are not considered a delivery system for the gene therapy of cystic fibrosis, and the research focus has shifted to other vectors (Table 4).

In the following clinical trials (NCT00073463, NCT00004533), the functional sequence of the CFTR gene was delivered using a vector based on adeno-associated viruses (AAV), which, depending on the dose, provoked a less pronounced immune response and were better tolerated by patients (Plasschaert et al. 2024; Lomunova and Gershovich 2023; Davies et al. 2024). However, the effectiveness of this strategy also turned out to be insufficient, and one of the possible reasons for this may be the low ability of AAV to pass through the mucus layer to the surface of the epithelium (Lomunova and Gershovich 2023). The results of the studies reported insignificant expression of CFTR and weak suppression of the manifestations of this disease (Plasschaert et al. 2024; Lee et al. 2021; Lomunova and Gershovich 2023). In addition, AAVs were unable to provide long-term CFTR expression (Plasschaert et al. 2024; Parisi et al. 2025). The main disadvantage of these systems is their small packaging capacity (less than 4.6 kb), which makes it impossible to pack a complete copy of the CFTR gene along with regulatory sequences (Lee et al. 2021; Davies et al. 2024). Therefore, shortened variants of the CFTR gene (mini-genes) were created, which were placed in AAV vectors, but this approach proved ineffective against class I mutations (Plasschaert et al. 2024). Nevertheless, on March 29, 2022, a clinical trial (NCT05248230) of cystic fibrosis gene therapy using the 4D-710 vector based on AAV was launched, which is intended for the treatment of adult patients who are not suitable for CFTR modulators (Table 4) (Davies et al. 2024).

Currently, lentiviral vectors are considered the most promising viral delivery system (Davies et al. 2024). They have low immunogenic properties, but at the same time they provide a longer expression of the delivered genes compared to Ad and AAV and can transduce the stem cells of the respiratory tract epithelium. At the same time, lentiviral vectors have sufficiently high packaging capacity to deliver a complete copy of the CFTR gene to cells (Davies et al. 2024). However, lentiviruses are integrating viruses and can cause genome instability or even induce oncogenesis, i.e., they exhibit genotoxicity (Plasschaert et al. 2024; Davies et al. 2024). Therefore, non-integrating lentiviral vectors lacking viral integrase and third-generation self-inactivating (SIN) vectors without lentiviral tat and transcription control elements in long terminal repeats (3′-LTR) are being developed (Plasschaert et al. 2024; Davies et al. 2024). Despite the initially low immunogenicity, during the production of vectors, lentiviruses can capture the membrane proteins of the cell lines in which they were developed; this can increase the immunogenicity of the finished vector and requires additional control (Plasschaert et al. 2024; Davies et al. 2024). In 2024, a clinical trial of BI 3720931 (Lenticlair™ 1) (NCT06515002) was launched. Lenticlair™ is a gene-therapeutic drug based on a pseudotyped, third-generation self-inactivating lentiviral vector equipped with the Sendai virus F-protein and neuraminidase (rSIV.F/HN), which delivers codon-optimized and CpG-free cDNA encoding CFTR (soCFTR2) under the control of a hybrid promoter also without CpG (hCEF) (rSIV.F/HN-hCEF-soCFTR2) (Davies et al. 2024).

Non-viral delivery systems

Non-viral delivery systems are a promising alternative to viral vectors; they have the largest packaging capacity and weak immunogenicity and are relatively easy to manufacture (Plasschaert et al. 2024; Lee et al. 2021; Parisi et al. 2025; Butt et al. 2022). A significant disadvantage of non-viral vectors is the low efficiency of delivering genetic material to cells and the short period of expression of the delivered gene; however, due to low immunogenicity, gene therapy using non-viral vectors can be used repeatedly (Xia et al. 2019).

Liposomes were one of the first non-viral delivery systems for the gene therapy of cystic fibrosis. In 1995, clinical trials of cationic liposome-based gene therapy (DMRIE/DOPE) were initiated, in which copies of the CFTR (pGT-1) gene were delivered to cells of the nasal mucosa (NCT00004471) (Table 4). They demonstrated the possibility of partial restoration of CFTR-mediated transport of Cl^‒ ions, but the positive effects persisted for a short time; one of the reasons for this may be active endocytosis, leading to retention of trapped DNA in perinuclear vesicles (Plasschaert et al. 2024).

Antisense oligonucleotides

Antisense oligonucleotides (ASO) are synthetic RNA or DNA molecules, 12–30 nucleotides long, that interact with the target mRNA through the Watson–Crick binding and modulate post-transcriptional modification of mRNA and translation or expression of genes using cellular mechanisms of degradation of DNA/RNA and RNA/RNA duplexes (Kim and Krainer 2023; Gopi et al. 2022; Lopes-Pacheco 2020). According to the mechanism of action, antisense oligonucleotides are divided into steric-blocking ASOs, which prevent the binding of proteins or ribonucleoproteins (RNPs) that regulate post-transcriptional modification of the target RNA or translation, and Gapmer ASOs, which suppress the expression of target genes by RNase H-mediated degradation of the target mRNA duplex and ASO (Kim and Krainer 2023). ASO is a promising technology for the treatment of various diseases, from hereditary to viral. It is noteworthy that the first approved drug for the pathogenetic treatment of spinal muscular atrophy (SMA) was ASO—nusinersen (Spinraza®). The success of ASO against SMA has prompted researchers to develop similar drugs against other genetic diseases and, in particular, cystic fibrosis.

Research is actively underway to create ASO targeting nonsense mutations, splicing mutations and capable of increasing CFTR expression in patients with these rare variants; in addition, ASO complementary to short open reading frames (uORFs) increase the expression and activity of CFTR with mutations F508del and N1303K (Kim and Krainer 2023; Lopes-Pacheco 2020). Nonsense mutations such as W1282X account for approximately 8% of cystic fibrosis-causing CFTR gene mutations; their pathogenesis is based on nonsense-mediated mRNA degradation (NMD), the targeted inhibition (gene-specific antisense inhibition of NMD, GAIN) of which for CFTR mRNAs can potentially increase the amount of CFTR, and thereby spread the effect of CFTR modulators for the W1282X mutation and some other class I mutations in combination therapy (Kim and Krainer 2023; Lopes-Pacheco 2020). Antisense oligonucleotides targeting defective splicing sites restore splicing and increase the amount of the normal isoform of CFTR mRNA, suppressing the inclusion of pseudoexones in mRNA in the presence of mutations such as 3849 + 10kbC > T (Duguépéroux and Braekeleer 2005; Kerem et al. 2021; Kim and Krainer 2023; Lopes-Pacheco 2020). However, as in the case of vectors, the effective delivery of ASO to lung epithelial cells remains an unresolved problem, and ASO may also exhibit toxicity. But researchers are actively working to solve these problems, and there are drugs for the treatment of cystic fibrosis based on ASO that have reached clinical trials. In 2015, two clinical trials of QR-010, from ProQR Therapeutics, were launched (Table 4). QR-010, now known as eluforsen, is a 33-nucleotide long ASO RNA with a phosphorothioate backbone (PS) and complete 2′-O-methylation (2′-OMe), which, by an unknown mechanism, binds to CFTR mRNA in the region of three-nucleotide deletion, leading to loss of phenylalanine at position 508 in patients with the F508del mutation (Lomunova and Gershovich 2023; Kim and Krainer 2023; Sermet-Gaudelus et al. 2019; Drevinek et al. 2020). According to the results of these studies, eluforsen was recognized as safe; it was found that it is able to restore CFTR function (NCT02564354) and improve the quality of life of patients, but its effectiveness was low. There was no significant improvement in lung function and chloride content in sweat compared with placebo (NCT02532764) (Kim and Krainer 2023; Sermet-Gaudelus et al. 2019; Drevinek et al. 2020). In 2022, the development of eluforsen was discontinued (Kim and Krainer 2023; Lopes-Pacheco 2020).

Genome editing

Genome editing is a field of gene therapy that includes methods for purposefully altering the nucleotide sequence of genes using “programmable” nucleases and their modifications. Direct correction of mutations can potentially guarantee the lifelong elimination of manifestations of monogenic diseases and, in particular, cystic fibrosis; however, there are serious obstacles to their use, preventing the creation of safe and effective therapy at this stage of the development of genetic technologies in medicine. However, research in this direction is actively underway using various genome editing tools. There are high hopes for this direction, since it is devoid of such disadvantages of gene replacement therapy as the induction of non-physiological expression of the delivered gene due to the action of a foreign, most often viral, promoter, as well as a high risk of loss of the transgene located in the nucleus in the form of an episome (Maule et al. 2020).

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALEN)

Attempts to directly correct mutations in cystic fibrosis have been made since 2008, when various scientific groups used zinc finger nucleases (ZFNs) to correct the F508del mutation in vitro (Maeder et al. 2008; Lee et al. 2012). ZFNs are synthetic zinc—containing endonucleases capable of introducing double-stranded breaks (DSB) in a specific DNA region. They consist of two parts: the actual “zinc fingers” (ZF) are several domains that function as a DNA-binding unit and determine the specificity of ZFNs and nucleases of FokI— a catalytic unit (Maeder et al. 2008; Lee et al. 2012; Katayama et al. 2024). Each ZF recognizes a three-nucleotide DNA sequence, so ready-made ZFN dimers consisting of three tandem ZF domains recognize 18-nucleotide binding sites in the target DNA region (Lee et al. 2012; Katayama et al. 2024). For each task, it is necessary to design new ZFNs capable of binding to the target sequence, which complicates the development and production process, but at the same time, ZFN cDNAs have a smaller size (0.3–0.6 kbp) compared to other genomic editors such as TALEN (1.7–3 kbp) and Cas9 (4.1 kbp); therefore, they are easier to package into a viral vector (Lee et al. 2021; Katayama et al. 2024). However, attempts to correct the F508del mutation using ZFNs by inserting a short DNA fragment have shown very low (less than 1%) repair efficiency. Therefore, other genomic editors were used in further studies (Lee et al. 2012).

In later studies, transcription activator-like effector nucleases (TALEN) were used to develop gene therapy for cystic fibrosis. The TALEN system, created in 2011, is structurally similar to ZFNs and consists of TALE repeats responsible for recognizing the target DNA region and the FokI nuclease, which introduces a double-stranded break (Lee et al. 2021; Xia et al. 2019). The main advantage of TALEN over ZFNs is greater binding specificity due to the recognition of sites of 34 base pairs, which is even more than that of the CRISPR/Cas9 system, which recognizes about 20 base pairs, which facilitates gene editing primarily through homologous recombination (HDR) and reduces the likelihood of inappropriate editing (Lee et al. 2021; Xia et al. 2019).

From 2016 to 2019, TALEN was used to correct the F508del mutation in cultures of induced pluripotent stem cells (iPSCs) obtained from patients with cystic fibrosis (Xia et al. 2019; Suzuki et al. 2016; Merkert et al. 2017). In 2017, IPSC culture (MHHi002-A) obtained from peripheral blood endothelial colony-forming cells (ECFC) was transformed by embedding single-stranded DNA with a length of 105 base pairs (ssODN) into the CFTR gene with this mutation to include the missing “CTT” trinucleotide, but the editing efficiency turned out to be extremely low (Merkert et al. 2017). In a 2016 study, IPSC culture of epithelial cells of the submucosal glands of the respiratory tract (CFSME101) of a patient with the F508del mutation in the homozygous state was transformed with short DNA fragments 491 bp long (SDF wtCFTR, 491z-SDF), encoding normal CFTR and TALEN expression vectors. Allele-specific analysis of the CFTR gene mRNA expression level by RT-PCR revealed both wild-type edited CFTR mRNA and F508del mRNA, and no inappropriate editing was detected, but the efficiency of mutation editing after six enrichment cycles, which increased the number of edited cells by 100 times, was no more than 10% (Suzuki et al. 2016). In 2019, the IB3-1 cell line obtained from bronchial epithelial cells of a cystic fibrosis patient with F508del and W1282X mutations was immortalized using a hybrid of adenovirus 12 and SV40 virus (Ad12SV40), and the cDNA of the CFTR mini-gene was packaged into helper-dependent adenoviral vectors (HD-Ad). Together with TALEN cDNA, lacZ reporter gene, and EGFP gene, according to these experimental results, the efficiency of integrating an 8-kb genetic construct into the target AAVS1 locus was about 5% (Xia et al. 2019). However, the study by this group has shown the possibility of delivering genes to progenitor cells, which in the future can be used to create gene therapy that ensures lifelong transgene expression. In addition, there were no serious prerequisites for the occurrence of immune reactions against edited cells (Xia et al. 2019).

CRIPSR/Cas9

CRISPR/Cas (clustered regularly interspaced short palindromic repeats and associated proteins Cas, CRISPR-associated) is a system of adaptive immunity of some bacteria and archaea, the mechanism of action of which includes three stages (Navarro et al. 2025):

1) Adaptation, which consists in the inclusion of new spacers in the CRISPR locus, which cut out the Cas1 and Cas2 proteins from a region of genetic material containing PAM (protospacer adjacent motif) that has penetrated the cell.

2) crRNA expression, which includes transcription of the CRISPR locus in the form of a long pre-crRNA, which is then processed by ribonucleases, the mechanism of which differs in different types of CRISPR/Cas systems.

3) Interference of nucleic acids homologous to the spacers contained in the CRISPR locus, in which crRNA complexes with Cas nucleases occur in the case of complementary binding of foreign DNA and crRNA. The interference mechanisms also differ between different types of CRISPR/Cas systems: types I, III, and IV use complexes of several Cas proteins, while types II, V, and VI use separate Cas proteins.

In 1987, CRISPR locus were discovered in the 3′-flanking region of the Escherichia coli iap gene, and in 2012 the CRISPR/Cas9 genomic editing system was created based on the Cas9 protein of the CRISPR/Cas type II system of the Streptococcus pyogenes and synthetic chimeric guide RNA (sgRNA, single-guide RNA, gRNA, tracrRNA:crRNA) combining trans-activating RNA (trans-acting RNA, tracrRNA) and crRNA (Maule et al. 2020; Parisi et al. 2025; Wang 2023; Jinek et al. 2012). This discovery has revolutionized the field of genetic technology. The advantages of CRISPR/Cas are versatility, high specificity, and low cost; and due to the simple design of Cas endonucleases, it was possible to create many tools based on this system, not only for gene editing, but also for diagnostics (Maule et al. 2020). However, there are several fundamental limitations to the use of CRISPR/Cas technologies in clinical practice, including high risk of inappropriate editing; immune reactions to various components of the system, including the vector and the Cas protein; difficulties associated with the delivery of system components to target cells in vivo (Navarro et al. 2025). Nevertheless, the fundamental limitation of using CRISPR/Cas9 technology for in vivo gene therapy of cystic fibrosis is the low frequency of precise editing by homology-directed repair (HDR) in respiratory tract epithelial cells; instead, editing by non-homologous end joining (NHEJ) prevails, but ways to overcome it are being actively developed (Graham and Hart 2021; Kantor et al. 2020).

CRISPR/Cas technology is considered the most promising system for creating gene therapy against monogenic diseases, various strategies for correcting mutations are being selected, and vectors and Cas proteins are selected and modified. Preclinical studies on cell lines and animal models show the effectiveness of using the CRISPR/Cas system to correct mutations in the CFTR gene, which indicates the realism of this approach to creating a gene therapy for cystic fibrosis.

Gene editing strategies using the CRISPR/Cas system to correct mutations in cystic fibrosis are being actively developed. Currently, the most promising strategies are based on homologous repair (HDR), non-homologous end joining (NHEJ), correction of point mutations using base editors, as well as prime editing (Maule et al. 2020).

The strategy of editing genes using HDR was developed immediately after the creation of the CRISPR/Cas system and is now preferred due to its high accuracy; but at this stage of the development of genetic technologies, its effectiveness is insufficient, since the restoration of double-stranded breaks by HDR occurs quite rarely (Maule et al. 2020). The first work in this area was published in 2013, when CRISPR/Cas9 was used to correct the F508del mutation in primary cultured small (SI) and large (LI) intestinal stem cells obtained from patients with the homozygous F508del mutation (Maule et al. 2020; Wang 2023; Schwank et al. 2013). The edited stem cells were able to produce organoids that exhibit physiological activity in response to the addition of forskolin, in which the CFTR channels were restored (Schwank et al. 2013). In 2015, IPSC culture obtained from skin fibroblasts of patients with the homozygous F508del mutation was transformed by a vector encoding the CFTR gene using CRISPR/Cas9 and sgRNA targeting sequences in the region of the F508del mutation (Wang 2023; Firth et al. 2015). The edited iPSCs were able to differentiate into epithelial cells of the respiratory tract, in which CFTR functions were restored, while inappropriate editing was not detected (Firth et al. 2015). In 2019, the F508del mutation in upper respiratory tract basal stem cells (UABCs) obtained from cystic fibrosis patients was corrected using the CRISPR/Cas9 system delivered by the AAV6 vector achieving 30–50% efficiency and 20–50% restoration of CFTR function. The edited cells were successfully integrated into the submucosal membrane of the pig small intestine (pSIS), where they retained the ability to differentiate. These results have the potential to provide a strategy for ex vivo gene therapy of cystic fibrosis through autologous transplantation of respiratory tract stem cells with a corrected mutation in the CFTR gene (Vaidyanathan et al. 2020). In 2022, it was possible to embed a full-length cDNA of the CFTR gene into the genome of basal stem cells of the upper respiratory tract (UABCs) and human bronchial epithelial cells (HBECs) at the corresponding locus using CRISPR/Cas9 and two adeno-associated viruses, each carrying its own half of the CFTR gene, while restoring CFTR function by more than 70% of the indicator of control samples (Vaidyanathan et al. 2022).

Gene correction using non-homologous end joining (NHEJ) is used to knock out genes caused by the formation of indels and deactivate regulatory elements of the genome, such as elements regulating intronic splicing like intronic splicing silencers (ISSs) (Maule et al. 2020; Sanz et al. 2017; Vilà-González et al. 2024). Mutations that disrupt mRNA splicing of the CFTR gene occur in 10% of patients; in such cases pseudoexones containing premature stop codons are formed: c.1679 +  1634 A > G, which generates a strong splicing acceptor in intron 12, which is why 99% of transcripts contain a pseudoexone 49 nucleotides long with a stop codon “TAA,” and c.3718-2477C > T, which in intron 22 also creates a pseudoexone with the stop codon “TAA,” or an elongation of an existing exon at the 5′ end c.3140-26A > G (3272-26A > G) (Sanz et al. 2017). It has been shown that CRISPR/Cas9-mediated deletion of these mutations effectively eliminates splicing defects in vitro (Sanz et al. 2017).

Base editors (BE) are a modification of the CRISPR/Cas9 system that allows single-nucleotide substitutions to be made in the target DNA region by a deamination reaction, without using donor DNA and making double-stranded breaks (Maule et al. 2020; Kantor et al. 2020; Lahr et al. 2023). BE consists of cross-linked to each other deaminase, which performs base conversion by deamination, and nickase Cas9 (nCas9), which provides binding of the target sequence and introduces a single-stranded break into the complementary to sgRNA DNA chain, which triggers the repair process, or dead Cas9 (dCas9), a mutant variant of this protein that is unable to cut DNA (Wang 2023; Kantor et al. 2020; Lahr et al. 2023). Base editors are divided into two classes: cytosine base editors (CBE), which convert C to U, which is replaced by T during replication or repair, and adenine base editors (ABE), which convert A to inosine, which is replaced by G (Wang 2023; Kantor et al. 2020; Lahr et al. 2023). Base editing is considered a safer strategy of genetic engineering, since it does not involve the formation of double-stranded breaks and does not require the delivery of exogenous DNA, which reduces the likelihood of serious damage to genetic material due to inappropriate editing, translocations, and the formation of indels in coding and regulatory sequences (Maule et al. 2020; Lahr et al. 2023). SNPs are the cause of many genetic diseases. It is noteworthy that 67% of mutations leading to cystic fibrosis belong to them, and 15% of them can potentially be corrected by CBE and 46% by ABE (Krishnamurthy et al. 2021). For example, in 2021, the mutations leading to the formation of a premature stop codon (R553X and W1282X) and the mutation disrupting splicing (3849 + 10 kb C > T) have been fixed using the adenine base editor (ABE7.10max-SpCas9-NG). It was done in the CuFi-3 cell line obtained from a patient with mutations F508del and R553X and primary epithelial cells of the respiratory tract obtained from patients with mutations W1282X and CFTR-del2,3. DNA sequencing confirmed an average of 60% correction efficiency, and restoration of CFTR channel activity was also observed (Krishnamurthy et al. 2021).

Prime editing is a newly created tool based on CRISPR/Cas technology for high-precision genomic editing without the use of donor DNA and the formation of double-stranded breaks, through reverse transcription of the sequence at the 3′-end of pegRNA and allowing all known modifications to be introduced into the target DNA regions (Wang 2023; Kantor et al. 2020; Yan et al. 2024; Bulcaen et al. 2024). The prime editors consist of the Cas9 nickase, which forms a complex with a guide RNA (gRNA) and a prime editing guide RNA (pegRNA) with attached modified reverse transcriptase (RT) (Maule et al. 2020; Wang 2023; Bulcaen et al. 2024; Sousa et al. 2025). In the study from 2024, L227R and N1303K mutations that cause cystic fibrosis and cannot be treated with available modulators were corrected using prime-editing technique (Bulcaen et al. 2024). As the result, CFTR function and glycosylation were restored in the cell lines 3HA-L227R-HEK293T, 3HA-N1303K-CFTR HEK293T, and 16HBE with 25–30% efficiency. In addition, they were able to transduce intestinal organoids obtained from primary cells of patients homozygous for L227R and N1303K with lentiviral vectors encoding the prime editor, pegRNA and gRNA. While CFTR function was also successfully restored, both organoids exhibited forskolin-induced swelling (FIS); however, sequencing revealed rare indels in some organoids (Bulcaen et al. 2024). In 2025, a prime-editing system, including a modified prime editor and pegRNA (epegRNAs) with a pseudoknot motif at the 3′-end to protect against exonucleases, was used to correct the F508del mutation: in primary epithelial cells of the respiratory tract of patients with cystic fibrosis, the correction efficiency was 25%, and in the model of immortalized bronchial human epithelium, up to 58% (Sousa et al. 2025).

Editing of individual bases and prime editing are very promising methods for correcting mutations in extended genes, which cannot be replaced with a wild-type copy using a vector due to limited packaging capacity, and these methods also cause less inappropriate editing (Kantor et al. 2020; Bulcaen et al. 2024; Sousa et al. 2025). However, the transition from preclinical research to practical application and development of gene therapy drugs is difficult and fraught with difficulties associated with the effective delivery of components of this system to the corresponding lung cells (Parisi et al. 2025; Wang 2023).

Discussion

Over the past 30 years, significant progress has been made in understanding the etiology and pathogenesis of cystic fibrosis: pathogenetic therapy using CFTR modulators has been developed, and supportive therapy has significantly improved. Unfortunately, modern pathogenetic therapy is not universal: class I mutations associated with the complete absence of CFTR protein synthesis are almost impossible to correct even with the most modern modulators, and existing drugs are not suitable for all patients. In addition, CFTR modulators are not able to provide a long-term effect and get rid of the cause of the disease—mutations—so patients are forced to take expensive drugs throughout their lives. Cystic fibrosis remains a fatal and incurable disease that affects thousands of people worldwide, and the high price of modulators restricts access to pathogenetic therapy and places a serious burden on the healthcare system.

Therefore, it is necessary to develop new methods of more effective pathogenetic therapy that eliminates the direct cause of cystic fibrosis. Genetic engineering approaches such as gene replacement therapy and genome editing are aimed at correcting any mutations and can provide full recovery in the future (Caplen et al. 1995; Koshechkin et al. 2022). Gene replacement therapy, which consists in transferring a functional copy of a gene or its mRNA to ensure the synthesis of its product, is the earliest approach based on genetic technologies to create therapy against cystic fibrosis, and the main research in this direction is aimed at selecting and improving delivery systems. Some promising developments are currently undergoing clinical trials. Viral vectors able to deliver genetic material into cells more efficiently than non-viral ones, but they have a smaller packaging capacity and can often trigger an immune response. In addition, ASO are promising therapeutic agents capable of extending the effect of CFTR modulators on nonsense mutations and splicing mutations. The main developments in the field of genome editing are currently at the stage of preclinical trials or in vitro studies and are aimed at improving editing systems and genomic editors. High hopes are pinned on CRISPR/Cas systems and their modifications, such as base editors and prime editing.

Despite advances in genetic technology, the development of gene therapy for cystic fibrosis remains an unresolved issue. And today there is not a single approved gene therapy drug. The most severe manifestations of cystic fibrosis are associated with impaired lung function, while it is easy to deliver gene therapy drugs to the lungs by inhalation, etc.; therefore, the epithelium of the respiratory tract is a preferred target. Difficulties in the development of gene therapy are mainly related to the imperfection of existing editors and the presence of a dense layer of thick and viscous mucus on the surface of the epithelium; moreover, the epithelium of the respiratory tract is constantly being updated, which prevents the preservation of edited cells. It was also found that CFTR-mediated secretion and absorption of Cl^‒ ions in the lungs are distributed among different cell types, so there is a problem in finding specific target cells for gene therapy of cystic fibrosis.

But despite the existing difficulties, genetic technologies are now actively developing, delivery systems and genomic editors are being improved, their accuracy and safety are being improved, new methods and systems are being developed, and models for testing them are being created (Anashkina et al. 2021). Therefore, there is reason to believe that the creation of an effective and safe gene therapy against cystic fibrosis is possible in the near future.

Author contribution

A.S. and Y.O. wrote the main manuscript text, A.S. prepared figures 1-7. All authors reviewed the manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.