Inhalable gene and RNA therapy for cystic fibrosis: perspectives and progress in clinical development

ABSTRACT

Small molecule modulators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) play a key role in cystic fibrosis (CF) management. Around 10% of people with CF (pwCF), however, are ineligible for modulator therapy. Inhalable CFTR gene and RNA therapies have the potential to return normal CFTR function to the lungs and provide a viable alternative therapy for all pwCF, regardless of mutation. Since 1993, 22 clinical trials of CF gene and RNA therapies based on viral and non-viral vectors have been undertaken, along with many more pre-clinical studies. To date, however, no single system has shown sufficient efficacy to warrant translation of this technology into the market. Nevertheless, novel gene carriers for CF treatment are continuously being developed, building upon the outcomes of prior clinical trials and knowledge about how the gene and RNA vectors behave in vivo. Given the suboptimal efficacy observed in humans over the last three decades, this review provides an objective overview of the progress and future of the inhaled delivery technologies that have been examined to date and the observed challenges and benefits of each. This study explored several databases, including PubMed, Scopus, and Google Scholar from January to November 2025.

Graphical Abstract

1. Introduction

Cystic fibrosis (CF) is a hereditary disorder that results from a defective gene encoding the CF transmembrane conductance regulator (CFTR), which facilitates the movement of chloride ions and water across epithelial cells [1]. CF is estimated to affect approximately 100,000 people worldwide, although recent evidence suggests this may be much higher. Notably, CF is more prevalent in the Caucasian population, affecting 1 in 3000 live births. The median life expectancy for people with CF (pwCF) born between 2020 and 2024 in the UK is predicted to be 66.2 years [2]. Functionally, abnormalities in CFTR cause an increase in the rheology, consistency and volume of mucus throughout the body, which leads to a variety of medical problems. In the lungs, thick mucus impairs mucociliary clearance, blocks airways and increases the risk of recurrent lung infections. Together, this induces local inflammation that destroys airway integrity, leading to bronchiectasis and ultimately respiratory failure. In the pancreas, CFTR dysfunction leads to fat and nutrient malabsorption that causes poor weight gain and steatorrhea in children. Further, destruction of the exocrine pancreas causes localized inflammation, fibrosis, and loss of acinar cells, which directly triggers β-cell loss and impairs the function of remaining islets, resulting in CF-related, insulin-dependent diabetes [3]. In addition, pwCF often suffer from infertility, chronic sinusitis and an increased risk of dehydration due to abnormal sweat gland function as well as a myriad of other potential manifestations. To date, more than 2,000 CFTR mutations have been identified. Those that cause functional disease have been categorized into six classes based on pathogenic mechanisms and implications for CFTR protein integrity and function (Figure 1) [7,8]. Based on the traditional classification, classes I to III typically produce a more severe disease phenotype than other classes [9]. However, additional genetic factors can also affect the pathophysiology of CF, resulting in broad variations in the clinical manifestation of the same CFTR mutation between patients [8]. Regardless, knowing the specific mutation affecting each patient is a useful and widely used way of selecting (or personalizing) appropriate drug therapy options [10].

Figure 1.

CFTR mutation classes and the available therapy approaches (A) [4,5], and the mechanism of gene therapy for CF treatment (B) [6]. Created in Biorender. Munir, M. (2025) https://BioRender.com/m3q4xvj.

Conventional CF therapy has largely relied on symptom-based treatment, namely the provision of pancreatic enzymes and vitamins to make up for the digestive insufficiency in pwCF, airway clearance to delay lung disease progression, and antibiotic therapy to manage recurrent infections. This typically involves a grueling daily routine of physical and drug therapy that adversely affects the wellbeing of pwCF and their carers. The most recent advance in CF care has been the introduction of small molecule CFTR ‘modulators,’ which were first introduced onto the market in 2012 (Figure 1(A)). These drugs revolutionized CF therapy by being able to improve CFTR function to varying degrees in approximately 80–90% of patients and prolong life expectancy [11,12]. However, patients with mutations that are not receptive to modulator therapy still rely upon conventional management approaches. Further, the modulators can introduce adverse effects that can affect patient compliance and therapeutic responses to these drugs. These are discussed in detail later in this review [9].

The prospects for pwCF therefore remain uncertain, especially for those with a non- responsive mutation. For this reason, gene and RNA therapy has long been considered the ‘holy grail’ of CF management and is an area that has been gaining increased attention in recent years [13,14]. The objective of gene and RNA therapy is to introduce nucleic acids encoding wild-type CFTR into cells to restore normal CFTR function. This may be achieved in a number of different ways that are summarized in Figure 1(B). The main target for this form of therapy is the lungs since respiratory system dysfunction is the major source of CF-related morbidity and mortality. For this reason, much of the gene and RNA therapy field has focused on inhaled administration.

Despite the first clinical trial on CF gene therapy being conducted over 30 years ago (Table 1), no form of gene or RNA therapy has advanced beyond Phase IIb. The current review provides a critical overview of the progress made to develop a viable inhalable gene or RNA therapy for CF, and progress in clinical trials is summarized in Figure 2. We give an overview of the different systems that have been examined and compare their advantages and disadvantages to oral CFTR modulator therapy. In this review we utilized several databases, including PubMed, Scopus, and Google Scholar from January to November 2025, to identify relevant abstracts, reports, review articles, and research articles related to inhaled nucleic acid therapy to treat cystic fibrosis.

Table 1.

Clinical trials of inhaled gene and RNA therapy for cystic fibrosis from clinicaltrials.gov (initially accessed in December 2025).

Year (start/end)	Registration, trial phase, & sponsor	Study details	Delivery vector & genetic material	Administration method	Location (Patient Number)	Major outcomes	References
1993/NA	NCT00004779 Phase I	Phase I pilot study of Ad5-CB-CFTR in CF patients	rAd5, CFTR DNA	Intranasal instillation	US (12)	No results reported, and no follow-up to phase II	[15]
1995/NA	NCT00004287 Phase I	Phase I study of adenovirus H5.001CBCFTR in CF patients	rAd5, CFTR DNA	Bronchoscope-guided instillation	US (14)	No results reported, and no follow-up to phase II	[16]
1995/2001	NCT00004471 Phase I	Phase I pilot study of cationic liposome-mediated gene transfer in patients with CF	Liposome complex (DMRIE/DOPE), CFTR DNA	Intranasal instillation	US (9)	No results reported, and no follow-up to phase II	NA
1995/2002	NCT00004806 Phase I	Phase I study of liposome-mediated gene transfer in patients with CF	Liposome, CFTR DNA	Intranasal instillation	NA (9)	No results reported, and no follow-up to phase II	NA
1999/2002	NCT00004533 Phase I/II	Phase I randomized study of AAV-CFTR in patients with CF	rAAV2, CFTR DNA	Jet nebulization	US (19)	One pulmonary exacerbation was possibly vector-related, and DNA transfer was observed in the lung. The study proceeded to phase II	[17]
2003/2005	NCT00073463 Phase II/III (terminated at phase IIb)	Safety and efficacy of tgAAVCF in patients with CF	rAAV2, CFTR DNA	Instillation into the maxillary antrum	US (102)	Repeated administrations were deemed safe and well-tolerated; nonetheless, they did not result in significant improvements in pulmonary function.	[18–20]
2008/2010	NCT00789867 Phase I/II	Single dose of pGM169/GL67A in CF patients	Liposome, CFTR DNA	Breath-actuated jet nebulization	UK (35)	The examined drug was safe, evaluated through clinical exams, laboratory tests, imaging, lung function assessments, and immune and histology analyses. DNA transfer and electrophysiological responses were measured in airway samples.	[21,22]
2012/2014	NCT01621867 Phase IIb	Repeated application of gene therapy in CF patients	Liposome, CFTR DNA	Breath-actuated jet nebulization	UK (130)	A modest but significant treatment effect with evidence of stabilized lung function compared to the placebo group. No significant differences in treatment-related adverse events across groups. There was no follow-up to phase III	[23]
2015/2016	NCT02564354 Phase I (completed) (ProQR Therapeutics)	Exploratory study to evaluate QR-010 in subjects with CF (ΔF508 CFTR mutation)	Single-stranded RNA ASO in iso osmolar solution	Intranasal as an atomized liquid	US, Belgium,France (18)	QR-010 showed a favorable safety, but the treatment did not result in improved chloride or sodium transport NPD values in the heterozygous cohort (n = 7)	[24]
2018/2021	NCT03375047 Phase I/II (completed) (Translate Bio)	Evaluate the safety, tolerability of nebulized MRT5005 in adults with CF	LNP, CFTR mRNA	Unspecified nebulization	US (42)	MRT5005 showed favorable safety, except for febrile and hypersensitivity reactions. The treatment maintained the FEV1 stability, but no FEV1 improvement was observed.	[25]
2018/2020	NCT03647228 Phase I/IIa (completed) (Ionis Pharmaceuticals)	A phase I/IIa study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of single and multiple doses of IONIS-ENaCRx in healthy volunteers and patients with CF	ASO inhibitor targeting ENaC	Inhalation or nebulization	UK, Germany (98)	The treatment was well tolerated. Reduction in ENaC mRNA supports mechanistic efficacy at the doses and regimens tested, and supports further investigation of ION-827359 in pwCF	[26]
2020/2021	NCT04375514 Phase I/IIa (terminated) (Arrowhead Pharmaceuticals)	Study of ARO-ENaC in healthy volunteers and in patients with CF	siRNA targeting ENaC	Unspecified nebulization	Australia. NewZealand (43)	The study was terminated due to unexpected signals of local lung inflammation in a chronic toxicology study in rats.	[27]
2022/2024	NCT05095246 Phase I (withdrawn) (Krystal Biotech)	A Study of inhaled KB407 for the treatment of CF	HSV-1, two copies of CFTR DNA (a replication-incompetent and non-integrating vector)	Unspecified nebulization	Australia (0)	The study was withdrawn due to the COVID pandemic, logistical, and recruitment challenges.	[28]
2023/2025	NCT05504837 Phase I (ongoing) (Krystal Biotech)	A Study of inhaled KB407 for the treatment of CF	HSV-1, two copies of CFTR DNA (a replication-incompetent and non-integrating vector)	Unspecified nebulization	US (12)	The delivery system was mostly well tolerated by 7 patients with dose of 109 PFU via inhalation. An extensive airway distribution and epithelial cell transduction were observed in the airways.	[28,29].
2022/NA	NCT05248230 Phase I/II (ongoing) (4DMT)	4D-710 in adult CF patients with CFTR modulator therapy	rAAV-4D-A101, CFTR DNA	Unspecified nebulization	US (40)	The interim results demonstrated mild, but transient, adverse events, which were resolved by 2 months. At a dose of 2.5 × 1014 vg, durable transgene expression in airway cells was observed over 1 year, along with the improvements of ppFEV1, LCI2.5 and quality of life based on a 1-year questionnaire	[30].
2023/NA	NCT05668741 Phase I/II (recruiting) (Vertex Pharmaceuticals)	A Phase 1/2 Study of VX-522 in participants with CF (Class I mutation)	LNP, CFTR mRNA	Unspecified nebulization	US, Australia, Europe, UK (33)	The study is still recruiting participants.	NA
2023/2023	NCT06217952 Phase I (completed) (SpliSense)	Safety, tolerability, and pharmacokinetics of SPL84 in healthy volunteers	ASO targeting c.2657 + 5G > A (2789 + 5G > A) splicing mutation	Unspecified nebulization	Israel (32)	These successful results supported the initiation of a phase 1/2 clinical study of SPL84 (ongoing), assessing the safety, tolerability, and pharmacokinetics of a single ascending dose in healthy subjects to be followed by assessment of safety, tolerability, pharmacokinetics, and preliminary efficacy of multiple ascending doses in CF patients carrying the 3849 mutation.	[31]
2024/NA	NCT06429176 Phase IIa (recruiting) (SpliSense)	Safety, tolerability, pharmacokinetics, and preliminary efficacy of SPL84 in patients with CF (3849 + 10kb C->T mutation)	ASO targeting c.2657 + 5G > A (2789 + 5G > A) splicing mutation	Vibrating mesh nebulization	US (24)	The study is still recruiting participants.	[32]
2023/2024	NCT05712538 Phase I (completed) (Arcturus Therapeutics)	Safety, tolerability, and pharmacokinetics of ARCT-032 (LunairCF) in healthy adult subjects and adults with CF	LNP, CFTR mRNA	Unspecified nebulization	New Zealand (39)	ARCT-032 was safe and well tolerated, and the improvement of FEV1 was observed after 2 doses of ARCT-032.	[33]
2024/NA	NCT06515002 Phase I/II (withdrawn) (Boehringer Ingelheim)	A study to test how well BI 3,720,931 (Lenticlair) is tolerated and whether it improves lung function in people with CF (3849 + 10kb C->T mutation)	Third-generation lentiviral vector pseudo typed with Sendai virus F and HN envelope proteins	Unspecified nebulization	Europe, UK (36)	A press release in February 26 suggested that the therapy was well tolerated but the ‘clinical data did not support further investigation.’	[34]
2024/NA	NCT06526923 Phase I/II (recruiting) (Spirovant Sciences)	A phase I/II trial of SP-101 for the treatment of CF (SAAVe)	AAV2.5T, CFTR DNA (minigene)	Unspecified nebulization	US (15)	The study is still recruiting participants.	[35]
2024/NA	NCT06747858 Phase II (recruiting) (Arcturus Therapeutics)	Safety, tolerability and efficacy study of ARCT-032 in people with CF (LunairCF)	LNP, CFTR mRNA	Unspecified nebulization	US (12)	The study is still recruiting participants, but early data indicate that FEV1 is not significantly improved.	NA
2024/NA	NCT06237335 Phase I (recruiting) (ReCode Therapeutics)	A phase 1 study evaluating safety and tolerability of RCT2100 in healthy participants and in participants with CF	LNP, CFTR mRNA	Unspecified nebulization	UK (100)	The study is still recruiting participants.	[36]

Abbreviations: ASO: antisense oligonucleotide, DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DMRIE: 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide, ENaC: epithelial sodium channel, HSV-1: herpes-simplex virus 1, LCI_2.5: lung clearance index, LNP: lipid nanoparticle, rAAV: recombinant adeno-associated virus.

Figure 2.

Summary of the inhalable CF nucleic acid therapy clinical pipeline.

2. Delivery vectors for nucleic acids

To the best of our knowledge, the first clinical trial of gene therapy for CF treatment was reported in 1993 using CFTR cDNA delivered intranasally via an adenovirus. However, half of the reported clinical trials have only been undertaken in the last 5 years as shown in the clinical trial summary in Table 1. The number of pre-clinical investigations of novel viral and non-viral gene and RNA delivery systems undertaken has also steadily increased over the past three decades and these are shown in Table 2. Although many nucleotide-based therapies have been developed for CF, this review focuses on those that have reached the clinical phase, namely DNA, mRNA, siRNA and antisense oligonucleotides (ASO) (Table 1). The pre-clinical development of other nucleotides, including transfer RNA (tRNA), and gene editing methods has been reviewed elsewhere [6]. Readers interested in a comparison between the advantages and disadvantages of using DNA versus RNA-based therapies for CF are directed to section entitled ‘Nucleic Acids for CF Therapy: DNA vs RNA’ in the supplementary information.

Table 2.

In vivo pre-clinical studies of lung-delivered gene and RNA therapy for cystic fibrosis. Key results are summarized as positive (+) and adverse (-) outcomes.

Year	Delivery vector	Animal subject	Administration	Key results	Ref.
Viral vector
1994	AdCFTR	Adult rhesus monkey (M. mulata)	Intratracheal or intrabronchial administration	(+) No significant difference was observed in animals intranasally administered AdCFTR, followed by intrabronchial 24 hours later [2 ×107 to 5 × 1010 plaque-forming units (pfu)], only intrabronchial (1010 pfu), and vehicle controls. A dose-dependent increase was observed in inflammatory cells, mainly lymphocytes of the area where AdCFTR was administered, which lasted for at least 2 months. AdCFTR DNA was not detected in extrapulmonary organs.	[37]
1996	AAV-CFTR	Rhesus macaques	Intrabronchial administration to the posterior basal segment of the right lower lobe of the lungs	(+) The presence of vector mRNA was detected for 180 days post administration. No inflammation or other toxicity was observed in the lung but vector DNA was found in other organs of several subjects.	[38]
1999	rAAV-CFTR	Pasteurella-free NZW male rabbits	Instillation to the right lower lobe bronchus, along the middle portion of the posterior tracheal wall, and in the right nostril	(+) GFP expression was seen in the epithelial airway three weeks after injection, despite elevated anti-AAV neutralizing titers at the time of delivery. No notable inflammatory reactions were detected. These studies demonstrate that repeated airway delivery of rAAV facilitates safe and effective gene transfer, and that serum anti-AAV neutralizing antibody titers do not predict in vivo airway neutralization.	[39]
2002	rAAV2-GFP	Juvenile Rhesus Macaques	Direct administration to the tracheobronchial tree along with 99mTc-DTPA	(+) 99mTc-DTPA was uniformly identified throughout the lung, corresponding to an average dosage of 1.33 × 1010 ± 9.5 × 109 i.u. per area. The increased GFP expression was found when the dose exceeded 3 × 109 i.u.	[40]
2003	K18CFTR-HD-Ad	CD-1 mice, Cftr knockout mice (Cftrtm1UNC)	Small drops into nostrils	(+) CD-1 mice: 8. The CFTR was expressed in the airway epithelia of mice, and CFTR RNA and protein were found in whole lung and bronchioles, respectively, for 28 days after administration, while acute inflammation was minimal to moderate (+) KO mice: Mice receiving gene therapy prior to Burkholderia cepacia complex challenge demonstrated less severe histopathology and reduced lung bacteria level comparable to Cftr+/+ littermates	[41]
2003	AdlacZ	Female MF-1 mice	Intranasal administration	(+) Increased transgene expression (25-fold) was observed in the trachea and upper airways after 10-min pretreatment with 50 mM Na-caprate, corresponding to a 3-fold improvement over EGTA. In the more peripheral airways, expression of β-gal was increased 3-fold by Na-caprate while EGTA had no effect. The complex of AdlacZ and DEAE dextran raised the airway epithelia transduction after Na-caprate pretreatment was increased 45-fold over virus alone.	[42]
2005	HD-Ad-K18lacZ	SPF female New Zealand White rabbits	Intratracheal intubation	(+) Transgene was expressed in the large and small airways’ epithelium, from trachea to terminal bronchioles, particularly in the right lung. Transduction occurred in all surface epithelium cell types. Transduction of the epithelium was extensive, achieving 66% of cells in the trachea with virus formulated in isotonic 0.1% LPC. (‒) In contrast, virus formulated in 0.01% LPC resulted in a lower transduction rate of 24% in the trachea. A temporary reduction in dynamic lung compliance was noted immediately after aerosol administration. Additionally, mild-to-moderate patchy pneumonia without edema and fever were noted.	[43]
2012	F/HN-SIV-Lux, F/HN-SIV-GFP	Female C57BL/6N mice	Intranasal administration	(+) A lifetime of lung expression (∼2 yr) can be achieved by single dose administration, where a dose-related increase in gene expression was observed due to repeated daily administration. No chronic toxicity was observed during a 2-year study.	[44]
2015	PiggyBac/Ad or AAV	Female SCID mice	Intranasal administration	(+) Effective and sustained transgenic expression for up to eight weeks after nasal delivery of piggyBac/viral vectors in mice.	[45]
2016	AAV2H22-CFTR	CF pigs	Nebulization directly to airways using catheter	(+) Two weeks after administration, CFTR expression was observed in CF pigs, and a notable change in short-circuit current after stimulation with 3-isobutyl-1-methylxanthine and forskolin was detected in the paired right turbinate epithelia.	[46]
2016	FIV-CFTR	CF pigs	Intratracheal administration	(+) Pronounced increase in transepithelial cAMP-stimulated current in tracheal and bronchus tissues was observed, two weeks after treatment. Furthermore, increased pH in tracheal airway surface liquid and bacterial killing was also detected.	[47]
2017	rAAV2/HBoV1	Ferret	Intratracheal injection	(+) An efficient lung transduction can be achieved by rAAV2/HBoV1 in both newborn (3 to 7 days) and juvenile (29 days) ferrets, particularly in the distal airways. In addition, Transgene expression reduced with the growth of the ferrets post 7-day administration, and repeated dose at 29 days raised the transduction level (5-fold) compared to that observed in naive infected 29-day-old animals.	[48]
2018	HD-Ad-CMVGFP or HD-Ad-UBCLacZ	Female C57Bl/6 mice, Yorkshire female pigs	Intranasal delivery (mice) and direct bronchoscopic instillation (pig)	(+) Airway basal cells of mice and pigs can be targeted in vivo. CFTR gene can be delivered to airway basal cells from CF patients, and CFTR channel activity can be restored, three days after treatment.	[49]
2018	PiggyBac/Ad-GFP-T2A-gLuc	Wild-type and CF pigs	Intratracheal instillation	(+) Transduction in large and small airway epithelial cells of wild-type pigs was observed with GFP expression of ∼30–50% in surface epithelial cells, five days after treatment. The administration of piggyBac/Ad expressing CFTR resulted in phenotypically corrected CF pigs as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability.	[50]
2020	AAV2.5T-SP183-gLuc, AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc)	Neonatal and juvenile ferrets	Intratracheal administration	(+) gLuc activity peaked by five days after treatment and remained stable for up to fourteen days. (‒) Decreased transgene expression (11-fold), increased BALF NAbs, suppression of plasma anti-capsid-binding IgM merely in juvenile ferrets was observed after repeated dose administration. Notably, both age groups exhibited a reduced BALF anti-capsid binding IgG, IgM, and IgA antibodies post-repeated dose administration. Therefore, after repeated doses of AAV2.5T, age-dependent immune system maturation and isotype switching may influence the formation of high-affinity lung NAbs and offer a way to reduce lung AAV-neutralizing reactions.	[51]
2021	LV-V5-CFTR	Female and male CF KO rats (510X genotype)	Instillations into the right nostril	(+) LV-V5-CFTR treatment produced a mean correction of 46% towards wild-type chloride response in treated CF rats, seven days after treatment.	[52]
2021	LV-FLAG-Luc-GFP, LV-LacZ	Female and male Sprague Dawley rats	Intratracheal administration	(+) A week after treatment, LV-mediated airway gene transfer in the trachea was significantly improved by physical perturbation-based airway epithelium disruption techniques.	[53]
2024	rAAV2.5T-fCFTRΔR	Wild-type ferrets	Intratracheal administration	(+) Transgene expression declined (~5.6-fold) in 3 months after dosing and then stabilized for up to 5 months (~26% of control). rAAV NAbs in the plasma and BALF and ELISpot T cell responses to AAV capsid peptides were at the highest at 21-day and remained stable at 4–5 months after infection. Notably, compared to the first rAAV2.5T-fCFTRΔR administration, the second vector administration resulted in a 2.6-fold increase in the ELISpot T cell response and a roughly 2.3-fold decrease in fCFTRΔR mRNA and vector genomes, indicating that transduced cells from the first vector dosage were selectively destroyed.	[54]
2024	SP-101 (AAV2.5T)	G551D CF ferrets	Vibrating mesh nebulization	(+) SP-101 genomes were detected in the respiratory tract post single-dose administration of SP-101, followed by single-dose doxorubicin (an AAV transduction augmenter) or saline, and remained stable for up to 13 weeks. Antibodies that bound and neutralized the AAV2.5T capsid were seen irrespective of doxorubicin exposure. A T-cell response to AAV2.5T was poor in just a subset of ferrets, and no T-cell response to hCFTRΔR was observed.	[55]
Non-viral vector
1993	CFTR cDNA–Liposomes	CF mutant mice	Intratracheal instillation	(+) The gene therapy corrected the ion conductance defects in the trachea of CF mutant mice	[56]
1993	CFTR cDNA–liposomes	CF mutant mice	Jet nebulization or direct instillation to the intestinal tract	(+) Around 50% restoration of cAMP related chloride responses deficit in the CF mice, and modest correction in the intestine was observed four days after direct instillation. This study also provides a promising safety profile for both the airways and intestinal tract following the administration of liposome complexes.	[57]
1997	CFTR cDNA–liposomes	CF null mouse model (Cftrtm1Cam)	Intratracheal administration	(+) Double dose administration restored cAMP-stimulated chloride currents in trachea which were similar to normal mice or CF mice after single dose administration. A lung inflammatory response was not observed after double dose delivery.	[58]
1998	DODAC:DOPE NP (liposomes)	CD-1 mice	Intravenous or intratracheal administration	(+) Both administration methods demonstrated significantly higher transgenic expression compared to injection of free plasmid DNA. Such transgenic expression was at the peak 24 hours after administration and declined to ~10% of day-1 levels after two weeks. In addition, delivered DNA was detected in the distal lung region. Intratracheal administration mainly resulted in DNA deposition in epithelial cells of bronchioles, whereas intravenous administration led to DNA deposition in the alveolar region of the lung.	[59]
2010	Cationic lipid GL67A and MC or CMC	CF knockout mice	Nebulization in exposure chamber	(+) The increased mRNA transgene did not result in correction of the ion transport defects in the nasal epithelium of CF mice nor the immunohistochemical quantification of CFTR expression. Separated administration of CMC prior to DNA complexes did not increase transgene expression, whereas co-administration significantly increased gene transfer by 4-fold.	[60]
2011	Cationic liposomes (GL67A/pCIKCFTR)	Suffolk Cross ewes	nebulization	(+) Gene transfer and expression were observed 1 day after administration. Transgene mRNA was expressed in lung tissues, with median group values reaching 1–10% of normal CFTR mRNA. hCFTR was found in the epithelial cells of small airways in treated animals (two out of eight), and a mild local and systemic inflammatory response was detected.	[61]
2012	PEGylated polylysine compacted (CO)-DNA	CF knockout mice	Intranasal or intratracheal administration	(+) Although CO-CFTR showed a higher hCFTR protein level (9-fold) in HEK293 cells compared to normal cDNA, similar transient lung mRNA expression in normal and CF mice was observed after administration using nanoparticle complexes. A prolonged CO-CFTR mRNA expression (94, 71, 53, and 14% on day 2, 14, 30, and 59, respectively) was produced with the presence of novel prolonged expression element derived from the BGH gene 3′ flanking sequence.	[62]
2013	ENaCα siRNA (GSK2225745) LNP	Female BALB/c mice	Intranasal administration	(+) The treatment showed significant inhibition of the ENaCα expression in the lung of mice 72 hours after administration, revealing the potential of this agent as a novel inhaled therapeutic for cystic fibrosis.	[63]
2013	Cationic lipid GL67A/pGM169	Suffolk Cross lambs	aerosol exposure via an endotracheal tube	(+) No adverse effects on hematology, serum chemistry, lung function or histopathology were observed. Acute responses were detected related to bronchoalveolar cellularity (transient increased neutrophils and macrophage numbers) 1-day post-administration. No cumulative inflammatory effect or lung remodeling was detected with successive doses. In addition, pGM169-specific mRNA was found at day 1 post-administration as indication of successful gene delivery	[64]
2015	PLGA-PNA-DNA	F508del mutation on a fully backcrossed C57/BL6	Intranasal instillation	(+) Gene correction was observed in the nasal and lung tissue of CF mice, and the alteration in the potential difference assay was detected in the nasal epithelium, four days after treatment.	[65]
2018	siRNA liposome	Female C57Bl6 mice	Oropharyngeal instillation	(+) Approximately 30% ENaC silencing in the lung of CF mice (persisted for 7 days) was observed after a single dose of siRNA. An increased silencing (~50%) was achieved with three doses of siRNA.	[66]
2018	NP-cmRNAhCFTR	CF mice	Intratracheal (i.t.) spraying or intravenous (i.v.) injection	(+) The administration of chemically modified mRNA nanocomplexes (NP-cmRNAhCFTR) significantly improved forced expiratory volume in 1 s of CF mice, six days after treatment.	[67]
2018	LNP-cmCFTR	CF mice	LNP was pipetted onto the nostrils for spontaneous inhalation	(+) LNP-cmCFTR administration Cl-secretion to conductive airway epithelia via CFTR in CF mice for a minimum of 14 days. On day 3 post-administration, CFTR was at peak, restoring up to 55% of the net Cl− efflux of normal mice.	[68]
2019	RNA oligonucleotide (eluforsen)	FVB Cftrtmi1EUR mice	Intranasal instillation	(+) Systemic exposure was obtained after orotracheal administration in mice, and significantly increased CFTR-mediated saliva secretion was observed in female F508del-CFTR mice, which persisted for up to 13 days, and significantly decreased after 22 days. In addition, significant improvement of nasal potential difference and CFTR conductance were detected in two CF mice	[69]
2019	ASOs targeting ENaC subunit mRNAs	Non-genetic mouse model of ‘CF-like’ lung dysfunction	Orotracheal instillation	(+) There was a strong correlation between a decrease in amiloride-sensitive channel conductance and a decrease in ENaC subunits. Furthermore, reduced level of any ENaC subunit enhanced lung function and decreased levels of mucus indicators Gob5, AGR2, Muc5ac, and Muc5b, PAS reagent goblet cell staining, and neutrophil recruitment.	[70]
2019	peptide–poloxamine/SB transposon complex	B6CF mice	Intratracheal by microspray	(+) Peptide-poloxamine nanoparticles mediated RNA and DNA expression in the lung of CF mice (seven weeks) with negligible toxicity.	[71]
2020	α-ENaC subunit mRNA lipid NP	Cftr−/−tm1Unc Tg(FABPCFTR)1 Jaw/J double-transgenic CFTRKO mice	spontaneous inhalation into single nostril	(+) A decreased amiloride-sensitive nasal potential difference was observed after administration in CF mice for up to two weeks.	[72]
2020	Peptide coated PEGylated-NP	Balb/c mice	Intratracheal administration	(+) Peptide coating increased NP penetration through human CF mucus (~600-fold) and uptake in epithelial cells compared to non-coated NP. It also demonstrated enhanced homogenous distribution and retention in the mouse lung.	[73]
2022	PNA NP	Male and female mice homozygous for the F508del mutation	Intravenous administration via retro-orbital injection	(+) After intravenous administration, the study verified genotypic and phenotypic changes in F508del mice in vivo. As shown by in situ potential differences and Ussing chamber experiments, in vivo therapy led to a partial gain of CFTR function in epithelia, two weeks after treatment. Additionally, CFTR was corrected in both GI and airway tissues, with no off-target effects above baseline.	[74]
2024	HPAE/DNA CFTR polyplex	CF mouse model	Instillation and nebulization	(+) The nanocomplex polyplex exhibited excellent in vivo delivery efficacy in the lungs of CF mice.	[75]
2023	Cas9 mRNA, sgRNA, and donor ssDNA LNP	Homozygous G542X mice	Intravenous injection	(+) LNP administration successfully corrected the CFTR mutations in the mice and in homozygous F508del mutations in patient-derived human bronchial epithelial cells, which resulted in the restoration of chloride transport function and CFTR protein production, a week after treatment.	[76]

Abbreviations: BGH: bovine growth hormone, DODAC: dioctadecyl dimethylammonium chloride, DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, ENaC: epithelial sodium channels, HPAE: highly branched poly(β-amino ester), LNP: lipid nanoparticle, LPC: L-α-lysophosphatidylcholine, PAS: periodic acid-Schiff’s, PLGA: poly(lactic-co-glycolic acid), PNA: peptide nucleic acid.

Appropriate selection of the nucleic acid delivery vector plays a critical role in determining the efficacy and safety of the therapy since it dictates how the DNA or RNA cargo enters and traffics through cells, enters the nucleus and integrates into the chromosome if required, and expresses their information. Further, it is important to remember that the nanometer-sized delivery vector can also stimulate local inflammatory responses in the CF lung, and this is largely driven by physicochemical properties of the vector and interaction with immune and epithelial cells. During inhaled administration, delivery vectors also play an important role in navigating the nucleic acid cargo through the mucus barrier to access the underlying epithelial layer. To date, seven viral vectors and nine non-viral lipid-based formulations (liposomes and lipid nanoparticles) have been formally evaluated in clinical trials. Eight other trials were also conducted, but the identity of the carrier was not revealed. Several other delivery vectors are also being actively explored by research organizations and small companies, but have not yet reached the clinical trial stage. This section provides an overview of each delivery vector that has or is being investigated and their translational potential in light of their advantages and disadvantages, while the summary of CF gene therapy in the clinical stages is presented in Figure 2.

2.1. Viral vectors

Virus-mediated transfer of nucleic acids is the oldest method of delivering genetic material in vivo to treat various diseases, including CF. In the last few decades, numerous viral vectors have been developed that have shown promising results in vitro and in vivo (Table 2), with several systems reaching clinical trials. For the most part, clinical trials have demonstrated reasonable safety, but to date, none of these systems have reached phase III trials due to a lack of significant efficacy [50,77].

The mechanism by which viral vectors enter cells typically occurs through attachment to specific cell surface receptors. These receptors are generally expressed on either the apical or basolateral side, as illustrated in Figure 3. Following attachment to the specific receptor, the viral vector with a lipid envelope enters cells through membrane fusion, while non-enveloped vectors penetrate the cellular membrane via receptor-mediated endocytosis [78].

Figure 3.

The structure and receptor of viral vectors used for CF gene therapy and their cellular uptake pathways for CF treatment. Created in BioRender. Munir, M. (2025) https://BioRender.com/9gktcrz.

To date, four types of viral vectors have been investigated for CF nucleic acid therapy in clinical trials, including adenovirus, adeno-associated virus (AAV), herpes simplex virus-1 (HSV-1), and lentivirus. As discussed below, viral engineering has the distinct potential to produce ‘CF-tailored’ viral vectors that exhibit defined cellular targets to enhance efficacy and potentially, safety.

2.1.1. Adenovirus

Adenovirus was the first viral vector approved for clinical use in 2004 for cancer gene therapy. They exhibit an icosahedral morphology, characterized by an external protein shell encasing an internal nucleoprotein core, with diameters ranging from 70 to 100 nm [79,80]. After entering cells via the coxsackie and adenovirus receptor (CAR), that is mainly located on the basolateral side [80], the partially disassembled adenovirus is guided by motor proteins like dynein to the nuclear pore complex (NPC). This is followed by DNA release from the capsid and penetration into the nucleus as episomal DNA which is transiently expressed without integrating with the host’s DNA [81]. In this regard, adenovirus vectors do not have the capacity to stimulate insertional mutagenesis and potential risk of cancer.

Adenovirus was the first vector used in clinical trials for CF gene therapy and was administered via instillation into the nasal epithelium. Although the presence of the vector and gene transfer was detected, less than 1% of the patients’ epithelium was transduced 4 days after delivery and no significant restoration of chloride transport was observed. In addition, mucosal inflammation was found in two of three patients given the highest dose (2 × 10¹⁰ pfu), while there were no adverse events observed at the lower doses (2 × 10⁷ to 2  × 10⁹) [15]. Similar results were also obtained when the adenoviral vector was administered to the lungs via bronchoscopy-guided instillation. The gene was undetectable in cells after 43 days [16].

As a result of the lack of efficacy for the adenovirus-based delivery system, these vectors have not been examined further as an inhaled therapy for CF (Table 1). The poor performance of this system was likely due to the predominant basolateral tight junction location of the receptor coupled with difficulty in penetrating the muco-epithelial barrier of the lungs (Figure 3) [82]. However, improved access to the basolateral surface may be possible by employing methods to disrupt tight junctions, such as the local application of a saline solution [83]. Significant immune-related adverse effects related to the induction of adenovirus-specific cell-mediated T cells have also been a notable problem in clinical trials [16]. Collectively, therefore, adenoviruses are not an appropriate nucleic acid vector for inhaled delivery.

2.1.2. Adeno-associated virus (AAV)

To improve upon the lack of efficacy and insufficient safety profile exhibited by adenoviral vectors, adeno-associated virus (AAV) was investigated as an alternative non-integrating delivery vector. The first AAV gene therapy approved for market was Glybera for lipoprotein lipase deficiency in 2012. Since then, six other AAV-based injectable therapies have been approved, making it the most widely employed systemic gene delivery vector in the clinic. AAVs are a naturally replication-defective single-stranded DNA parvoviruses that are significantly smaller than adenoviruses (25 nm). This does, however, somewhat limit their nucleic acid loading capacity when compared to adenovirus.

In comparison to the adenovirus-based system, AAVs have demonstrated significantly better safety after three clinical trials in pwCF with a collective total of 102 participants. This is indicated by the absence of adverse respiratory issues, enhanced inflammation, and increases in serum-neutralizing antibody titer to AAV. Improved safety of AAVs was expected since they exhibit lower, but not insignificant, immunogenicity, which is discussed further below. However, efficacy was still a notable problem, and while tgAAVCF DNA was found in 80% of sinus specimens notable sinus transepithelial potential difference was not [18–20]. Like adenovirus, AAVs primarily enter cells via endocytosis through the basolateral membrane (KIAA0319L receptor). This can limit their transduction capability following inhalation. AAVs, however, can also utilize a combination of receptors (such as glycans and integrins) and endocytic pathways (including clathrin-mediated endocytosis, micropinocytosis, and a clathrin-independent pathway) to facilitate cellular entry. Endosomal escape also utilizes an acidic environment-activated PLA2 domain that functions like a phospholipase to enhance membrane disruption. Due to their smaller size, AAV then have a better capacity to enter the nucleus without requiring initial capsid disintegration [84].

In terms of capsid development, directed evolution of AAV plays an important role in the development of these gene therapy vectors. This technique simulates natural evolution by generating a broad array of biomolecules via various methods, such as random point mutagenesis, and employing repeated functional selection to produce variants with defined characteristics. As an example, directed evolution was recently utilized in the development of 4D-710 which entered phase I/II clinical trials in 2022 (NCT05248230) [85]. For a more extensive review on this area the reader is directed to the following review [86].

Another attempt to enhance the efficacy of AAV-based gene therapy was undertaken by using CFTR with a partially deleted R domain. This was expected to be beneficial for AAVs that exhibit a limited loading capacity. The opening of the CFTR channel requires phosphorylation of the serine residues at the R domain by cAMP-dependent protein kinase. The partial removal of the R domain (ΔR(708–835)-CFTR) leads to the generation of a protein kinase A phosphorylation-independent chloride channel. This has an opening probability of around 30% compared to the wild-type CFTR channel. Despite this reduced function, the efficacy of delivering CFTR via an AAV was demonstrated by transducing the nasal epithelium of CF mice and restoring the Cl⁻ transport defect. An alternate R domain deletion (ΔR(708–759)-CFTR) could retain CFTR function in an in vitro study [87]. The pre-clinical potential of AAV-loaded partially deleted R domain CFTR has resulted in its entry into an ongoing phase I/II clinical trial (NCT05248230). By using partially deleted R domain CFTR, a small promoter and poly A sequence, such as a 100 bp enhancer combined with an 83 bp minimal promoter (SP183 enhancer-promoter sequence), can also be incorporated to improve CFTRΔR expression [88]. The combination of SP183 and CFTRΔR with AAV2.5T (a hybrid of AAV2 and AAV5 with a single A581T mutation) has ultimately resulted in the SP-101 gene delivery system that is currently in phase I/II clinical trials. SP-101 can transduce epithelial cells via the apical as well as basolateral surfaces to maximize CFTR expression [55]. Although this system has the potential for improved efficacy, and no significant adverse effects were noted during the pre-clinical stage, the application of this vector to the lungs of pwCF is not without risk. After systemic administration, AAVs increase the risk of genotoxicity, hepatotoxicity and thrombotic microangiopathy [89]. While these are unlikely to be significant risk factors after inhaled delivery due to the localization of the nano-sized therapy to the lungs, immune-related adverse effects are a potential problem. As mentioned above, AAVs are still recognized by the immune system and can activate AAV-specific B cells. Given that the majority of the population exhibits neutralizing antibodies to AAV2 and/or AAV5, coupled with the fact that the lungs have a highly active immune system, when inhaled AAVs could stimulate local inflammatory responses [89]. This could temporarily compromise lung function in pwCF and increase the risk of lung infections. While temporary immune suppression in the lungs may limit inflammatory reactions, this also increases the risk of lung infections. Further, AAV neutralizing antibodies could limit nucleic acid delivery and therefore efficacy, particularly as repeated administration would be required. Successful translation of inhaled AAV-based DNA and RNA therapies for pwCF care therefore hinges on the demonstration clinical benefits in the lungs, demonstrated by enhanced lung function and reduced pulmonary exacerbation, coupled with risk mitigation strategies to limit immune-related adverse effects.

Despite the safety risk related associated with the AAV vector, the interim results of a Phase II study of 4D-710, an AAV designed to penetrate lung mucus, demonstrated mild, but transient, adverse events which were resolved by 2 months. At a dose of 2.5 × 10¹⁴ vg, durable transgene expression in airway cells was observed over 1 year along with clinical improvements in ppFEV1, Lung Clearance Index (LCI_2.5) and quality of life based on a 1 year questionnaire [30]. Early clinical data therefore look promising.

2.1.3. Herpes simplex virus

To overcome the limited loading capacity and predominant basolateral entry point of AAVs, recombinant herpes simplex virus 1 (HSV-1)-based delivery vectors, which can carry two copies of CFTR DNA and a non-integrating episome, as presented in Table 1 (KB407), were developed to improve efficacy [90]. HSV-1 is a large (155–240 nm) spherical virus that contains an icosahedral capsid (125 nm) [91]. It primarily enters the epithelium through the apical side via interaction between viral glycoproteins B/C (gB/gC) and heparan sulfate proteoglycans (HSPGs) on the cell, allowing glycoprotein D (gD) to bind nectin-1 or nectin-2. This binding activates viral glycoprotein H/L (gH/gL) and enables gB, as a fusion protein, to facilitate the merger of viral and cell membranes and eventually release of the viral capsid and tegument into the cytoplasm. HSV-1 then utilizes the dynein motor complex and microtubular network to facilitate its trafficking toward the nucleus, followed by capsid docking to the NPC and delivery of the nucleic acid into the nucleus [92]. Although HSV is pathogenic, recombinant HSV-1 is replication-incompetent and non-integrating, improving its safety compared to native HSV [93].

Initial studies showed promising results, where similar levels of CFTR mRNA were detected in HSV-1-transduced CF-patient organoids when compared to airway organoids from healthy patients [25]. One HSV-based gene delivery system (KB407) has so far entered clinical trials in Australia (withdrawn due to pandemic, logistical, and recruitment challenges) and the US, where the interim results of a Phase I study was recently reported. As of January 2026, 7 patients had been administered 10⁹ PFU of KB407 via inhalation. The delivery system was mostly well tolerated by patients. While efficacy data are not yet available, extensive airway distribution and epithelial cell transduction were observed in participant airways [29]. A similar HSV-1 vector encoding IL-2 and IL-12 has recently been granted a Regenerative Medicine Advanced Therapy designation by the FDA to fast-track this inhalable gene therapy for lung cancers, having shown promising clinical results at a dose of 10⁹ PFU. While this gene therapy targets lung cancers which are easier to transduce compared to primary lung cells, it still offers hope for the CF community.

2.1.4. Lentivirus

The most recent inhaled viral gene delivery system being examined clinically for CF is a largely lentivirus-based system. Lentiviral vectors confer stable gene expression in cells and their progeny due to integration of the DNA into the host genome. They also have a lower propensity to induce inflammatory responses compared to the abovementioned viruses. Moreover, lentiviral vectors can be re-administered to the epithelium without a loss of efficacy [93]. To further expand tropism, increase cell penetration, and reduce immunogenicity, lentiviral vectors can be modified by envelope pseudotyping [94] To this end, several pseudotyped lentiviral vectors have been examined for CF gene and RNA therapy. Promising preclinical data have been demonstrated for lentiviral vectors pseudotyped with vesicular stomatitis virus G (VSV-G) [95] and GP64 [93] envelope proteins. As an example, after lung administration of VSV-G-pseudotyped lentivirus containing human CFTR DNA into marmosets, efficient transduction was detected in the lungs of five out of six animals without evidence of an inflammatory response [95]. GP64-pseudotyped lentivirus containing CFTR DNA was aerosolized into the lungs of three neonatal CF pigs. Two weeks later, a substantial amplification in transepithelial cAMP-induced current and elevations in tracheal airway surface liquid pH and microbial elimination were observed, suggesting efficient return-of-CFTR function. In addition, the acidified pH of airway surface liquid (ASL) in CF was normalized, resulting in the restoration of bacterial killing in the lungs [93,96]. However, neither of these systems have advanced to human clinical trials to date. To overcome the discussed limitations of inhaled virus-based gene and RNA therapy, a team led by researchers from Imperial College London strategically engineered a lentiviral vector-based gene therapy containing CFTR DNA (BI3720931). The rSIV.F/HN lentiviral vector was pseudotyped with Sendai virus envelope proteins F and HN to improve apical transduction of the airway epithelium [34]. rSIV.F/HN exhibits an HN protein that specifically binds to ciliated glycans – carbohydrate structures on the surface of cilia. This binding triggers a conformational change in the F protein, which mediates the endocytosis of LV.F/HN on the apical side [97]. To enhance the safety without reducing the gene transfer [98], accessory genes that are responsible for in vivo replication and pathogenesis (vif, vpr, vpu, and nef) [99] were removed [100]. Furthermore, the viral components were divided into many plasmid structures as an additional safety feature to prevent the theoretically potential formation of replication-competent lentiviruses [97]. Preclinical work in mice showed that up to 15% of airway epithelial cells were transduced after a single dose, which was predicted to be sufficient to produce a clinical benefit in patients. Importantly, cells continued to express CFTR for up to two years and repeated dosing was shown to be achievable without significant loss of efficacy [44,101]. Further, chronic toxicity was not observed during the 2-year follow-up period in mice [44]. Comparable results were achieved in a follow-up cynomolgus monkey preclinical study in which a single inhaled dose of BI3720931 was administered yielding an airway epithelial transduction efficiency of 9–12%. The expression of vector-specific mRNA was high without evidence of significant toxicity, but the specific cell type(s) transduced were not investigated [102].

At present, it is unclear which cell types critically need to be transduced with CFTR to return normal physiological function to the lungs. While it was previously believed that ciliated cells were an important source of CFTR [100], more recent work has shown that ionocytes and secretory cells are the most significant source of CFTR. There is also conflicting information in the literature regarding whether or not the transduction of ciliated cells can improve CF lung function. As an example, the transduction of 25% of human ciliated airway epithelial cells was previously shown to restore normal mucus transport [96], while a more recent study reported restored Cl⁻ secretion in CFTR transduced secretory cells but not in ciliated cells [102].

Nonetheless, because of the positive preclinical findings, BI3720931 (LENTICLAIR™) was advanced into human Phase I/II trials in 2024 by Boehringer Ingelheim (NCT06515002) [34]. Unfortunately, while well tolerated the clinical trial was discontinued by Boehringer Ingelheim in February 2026 since it ‘failed to produce clinical data supporting further development.’

2.2. Non-viral vectors

Despite the potential for the inhaled delivery of well-designed viral vector-based gene and RNA therapies to improve CFTR function in the lungs, these therapies are not without risk of significant adverse and long-term effects. Further, public perception of the use of viral vectors for gene and RNA therapy is highly varied [103]. Non-viral vectors in the form of nanoparticles (NP) have therefore also been examined for inhaled CF gene and RNA therapy. The main advantages of non-viral vectors are higher genomic payload, ease of production and a reduced potential to stimulate immune responses and genotoxicity [78]. The fact that NP provide a consistently non-integrating form of gene delivery and do not have defined receptors that may be located apically, basolaterally or both (at least for untargeted systems) also solves issues relating to the potential for genome mutations and a lack of accessible cell entry pathways. However, they rely heavily on the formation of electrostatic interactions with the cell membrane. The lack of genome integration also means that, as with other non-integrating viral vectors, repeat dosing is necessary as transfected cells are shed. While targeted NP for CF have been developed and examined at the preclinical stage, these more synthetically complex systems have not been advanced into human clinical trials [104,105].

To date, lipid-based nanoparticles (LNP) have been the most widely explored non-viral vector for CF, with nine clinical trials reported since 1995. The cellular uptake of DNA- and RNA-NP is mainly mediated by caveolin- or clathrin-mediated endocytosis, micropinocytosis and phagocytosis, depending on the size of the NP and cell-type, as illustrated in Figure 4. In addition, the increased surface presentation of cationic charge on the NP enhances electrostatic interactions with anionic charges on cell membranes to increase the potential for cell uptake. Although NP of any size can enter cells through a variety of different mechanisms, clathrin- and caveolin-mediated endocytosis tend to occur faster than other internalization pathways [109].

Figure 4.

Endocytosis and intracellular trafficking pathways of non-viral carriers [106–108]. Created in BioRender. Munir, M. (2025) https://BioRender.com/is5f5h7.

2.2.1. Lipid-based nanoparticle (LNP)

Of the different NP explored for biomedical applications, LNP have been the most successfully translated systems for applications ranging from drug delivery to vaccinations. Cationic liposomes are also widely used in research to facilitate nucleic acid transfection into cells. The size of LNP typically examined for this purpose is approximately 100 nm [68]. The earliest clinical trials that examined the respiratory transfection capability of cationic liposomes (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide [DMRIE], and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]) delivered via the nasal turbinate, however, revealed very little delivery of DNA [110]. To improve upon these disappointing outcomes, extensive preclinical screening was conducted to identify better liposomal gene delivery systems. This preclinical work resulted in the identification of an optimized carrier formulation for transfection, namely the cationic liposome GL67A made of a mixture of N4-spermine cholesteryl carbamate (GL67) – DOPE–1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) – polyethylene glycol 5000 (PEG5000). This formulation demonstrated better stability and enhanced endosomal escape [60,61,64]. At the same time, the use of human cytomegalovirus enhancer/elongation factor 1α sequence and modified EF1a promoter to control plasmid expression, and the elimination of unmethylated CG dinucleotide (CpG) from CFTR DNA delivered by GL67A resulted in enhanced in vivo transgene expression (≥56 days) and the absence of acute lung inflammation in BALB/c mice [111]. The nebulized administration of these systems to the lungs resulted in mild to moderate lung-related adverse effects, but also significantly improved lung function in the Phase I/IIa safety and efficacy study undertaken with 35 participants [21,22,64]. Subsequently, the Phase IIb clinical trial of pGM169/GL67A with dose administration at 28 ± 5-day intervals for 12 months to 130 participants resulted in improved FEV₁ (5% or more) compared to individual baseline values in 21 (18%) patients (6 placebo and 15 pGM169/GL67A groups). Although this study showed clinically meaningful benefit according to the European Medicine Agency’s perspective (specifically, an improvement or stabilization of lung function) absolute efficacy and consistency of response were not considered sufficient to warrant investigation in Phase III trials [23].

To enhance transfection efficiency, and therefore efficacy, CFTR mRNA was examined as an alternative to DNA since it can be directly translated into protein without needing to be translocated into the nucleus and transcribed. However, as discussed above, RNA is more susceptible to degradation compared to DNA [112]. Therefore, to enhance stability further refinement of the NP formulation or modification of the RNA is essential to sufficiently preserve RNA integrity in vivo [6]. MRT5005 is a biosynthetic mRNA with a 5’ cap and a poly A tail to enhance transfection efficiency and prevent degradation. The formulation of this mRNA into a protective LNP vector showed promising initial in vitro results, where CFTR activity was restored in Fischer rat thyroid gland cells cultured in chamber tests under polarizing conditions. Although comparable results were not seen in primary human bronchial epithelial cells obtained from pwCF, preclinical studies in healthy rodents and non-human primates demonstrated CFTR expression in the proximal and distal airways after lung delivery [113]. These favorable preclinical results led to MRT5005 being the first inhalable RNA-based system translated into a clinical trial for CF. In this trial, mild to moderate adverse effects were observed, with cough and headache being the most common effects reported. However, no significant and consistent beneficial effects were observed on FEV₁ in any of the dose groups [25].

Another LNP-based mRNA formulation, ARCT-032 (LunairCF), was examined in Phase I clinical trials. This study was divided into two parts; the first part involved 32 healthy adults administered a single nebulized dose, while the second one enrolled eight pwCF who received two nebulized doses. The adverse effects of ARCT-032 were generally considered mild and included temporary fever-like symptoms, such as a high temperature accompanied by headache, back or muscular discomfort and nausea, which could be mitigated by inhaled premedication with salbutamol. This Phase I trial was followed by a Phase II trial (NCT06747858) that is currently underway [33]. The interim results of this trial showed a lack of significant improvement in FEV1 after 28 days, despite a reduction in lung mucus in four of the six patients. FEV1, however, is not the primary endpoint. The trial therefore continues with 6 patients targeted to receive a 15 mg dose, followed by a planned 12-week safety and preliminary efficacy trial including up to 20 patients in the first half of 2026 [114].

Two other mRNA/LNP complexes recently entered Phase I/II clinical trials, namely VX-522 (NCT05668741) and RCT2100 (NCT06237335), developed by Moderna/Vertex Pharmaceuticals and ReCode Therapeutics, respectively. VX-522, however, was suspended in mid-2025 over ‘tolerability’ issues relating to the ascending dose aspect of the trial [115]. As of January 2026, the trial is listed as ‘Recruiting’ in clinicaltrial.gov.

In general, the clinical trials of lipid-based nanoparticles as non-viral vectors for DNA or RNA in CF treatment have demonstrated reasonable safety profiles with acceptable adverse effects. The main hurdle for the clinical application is ongoing inadequate efficacy due to low transfection efficiency, which is affected by cellular uptake and endosomal escape [116]. Further modification of the liposome formulation can potentially improve endosomal escape through membrane fusion or destabilization mechanisms, but this remains an ongoing issue [117]. Another challenge that LNP experience is penetration through the mucus barrier to access respiratory epithelial cells, which is more restricted for lipid-based substances due to hydrophobic interaction with mucus. One additional complexity to consider, however, is that while LNP can be easily modified to enhance mucus penetration using high-density PEGylation, as an example [118], the pharmacokinetics of LNP are also heavily dictated by the biological corona. While the impact of the protein corona on systemic circulation has been somewhat defined, this is less well described in the lungs, making it difficult to predict optimal surface properties for mucus migration, cell entry and endosomal escape [119]. At this point, it seems that viral delivery systems offer a clear advantage over LNP in terms of returning CFTR function to the lungs. While LNP are ‘safer’ in the lungs, adverse effects are still observed. However, this is not surprising given the inherently high immune responsiveness of the lungs to any nanometer-sized material, even though many lipids used in LNP mimic lung surfactants.

2.2.2. Polymeric nanoparticles

While polymer-based nanoparticles have also been widely explored for drug and nucleic acid delivery applications, they are not as widely represented in the clinic or clinical trials, especially for inhaled delivery. Inhalable LNP are also approved for clinical use, but no polymeric nanoparticles have reached this stage. Further, no inhalable polymer-based gene or RNA delivery systems have been examined clinically for CF treatment. They do, however, represent one of the most diverse classes of potentially inhalable drugs and nucleic acid carriers with the capacity to manufacture systems with a wide range of structural and physicochemical characteristics.

An important consideration with polymeric systems is that the cationic charge on scaffold materials must be carefully manipulated to balance effective incorporation of nucleic acids with cell binding affinity, while limiting cell cytotoxicity from excessive cationic surface charge [120]. Further, polymeric NP exhibit different cell uptake and trafficking characteristics compared to viral and lipid-based nucleic acid delivery systems. Specifically, viral vectors and LNP typically fuse or interact with endosomal membranes to induce endosomal disruption and cargo release (Figure 4). After the cell binding and active uptake of polymeric NP into endosomes and fusion with lysosomes, NP and their nucleic acid cargo can be degraded by lysosomal enzymes since they do not have an inherent ability to escape from endosomes. Polymeric NP must therefore be specifically designed with moieties to facilitate their endosomal escape [121].

One of the polymeric carriers explored for CF gene therapy is a peptide nucleic acid (PNA)-based NP with a PNA/DNA/PNA triplex structure. In situ potential differences and Ussing chamber assays have shown a modest increase in CFTR function in gastrointestinal and airway epithelia with no off-target effects after intravenous administration in mice homozygous for the F508del mutation [74]. Further modification of this NP was performed by substituting the gamma position with diethylene glycol. This improved DNA binding and enhanced CFTR function and gene editing by up to 32% based on genomic DNA analysis in primary nasal epithelial cells in mice [122].

To this end, cell-penetrating peptides (CPP) are widely employed as scaffolds on which to build polymeric nanoparticle-based nucleic acid delivery vectors. CPP are typically cationic peptides that can interact with negatively charged nucleic acids to form nanoparticles [123]. The earliest CPP (MPG, a 27 amino acid sequence) was inspired by the fusion of HIV gp41 and SV40 T-antigen sequences, facilitating cargo complexation and delivery into the nucleus. This CPP has been used to successfully deliver nucleic acids into cells via an endocytosis-free pathway which requires a relatively high CPP loading. Further refinement of CPP was therefore carried out to enhance cargo delivery efficiency through the endocytosis pathway, where the molecular mechanism is well-characterized and the endosomal escape strategy can be tailored [124]. During endosome acidification, the amphipathic α-helical secondary structure of CPP allows them to destabilize the endosomal membrane by inserting themselves into and rearranging the lipid bilayer (Figure 4) [125]. This ability is influenced by the arrangement of hydrophilic and hydrophobic amino acids [121,125] and the presence of branches in peptides [106]. Although no polymeric CPP-derived gene or RNA therapeutics have been explored in clinical trials, irrespective of the route of delivery, 35 clinical trials of injectable CPP-based nanoparticles as a carrier for small molecule drugs have been undertaken in the last two decades [126]. This supports the potential feasibility of polymer-based nucleic acid delivery systems, but a considerable amount of work is needed before their clinical utility as an inhalable system for CF can be thoroughly evaluated against viral and lipid systems. Below, we have therefore reviewed the in vitro and preclinical evidence for the utility of CPP-based nanoparticles as a nucleic acid delivery system for CF therapy.

The application of CPP in CF gene therapy has been largely as a coating agent for other NP to enhance mucus penetration and cellular uptake. Although CPP-coated NP show efficient mucus penetration in sputum collected from pwCF and can enter CuFi-1 cells, nucleic acid delivery and expression have not been evaluated [73]. CPP have also been modified with other compounds to improve gene delivery. For example, CPP-poloxamine NP have been shown to restore 40% of CFTR function in CF bronchial epithelial cells and induce long-term (over 2-months) CFTR expression in CF mice with negligible lung toxicity [71].

As a stand-alone gene and RNA delivery system, however, polymeric CPP based on histidine (H) and lysine (K) have been shown to effectively condense negatively charged nucleic acids, interact with the cellular membrane, and induce endosomal escape via the proton sponge effect. The idea to develop the histidine and lysine-rich (HK) peptide as a gene delivery system was first proposed by Midous and Mosigny who histidylated polylysine to give a degree of polymerization (DP) of 190. This increased the transfection capacity of the base polylysine by over 1000-fold [127].

Building on these results, Leng et al. reported that linear HK CPP is less efficient at delivering DNA compared to more branched HK CPP since branching induces the formation of more stable complexes with DNA and better induces endosomal escape. Further, the addition of histidine-rich tails to branched HK peptides can also increase transfection capability by up to 1000-fold since histidine enhances the buffering capacity of the peptide. However, the optimal degree of HK branching varies depending on the type of nucleic acid being carried, whereby 8-branched HK favors siRNA delivery, while 4 is sufficient for DNA delivery [106].

Branched HK-based polymeric peptides interspersed with other (proprietary) non-essential amino acids are currently being used as the basis of CF gene delivery technology that is being developed by Loxegen Holdings Pty Ltd (patent filed [128]). A key limitation of a simple CPP-based nucleic acid delivery system for inhalation, however, is the limited capacity to penetrate the mucus layer to access underlying cells [129–131]. For this reason, the Loxegen NP are coated in a layer of polyethylene glycol (PEG) to reduce particle adhesion to mucin fibers. In general, PEGylation reduces the positive charge of polymer-nucleic acid NP and increases particle size. Despite a potential adverse effect of increased particle size on mucus penetration/migration and lung retention can be improved by up to 70- and 4-fold, respectively, provided the particle size remains lower than the pore diameter in CF mucus, which can range from 110 to 930 nm [132]. Mucus penetration can still, however, be enhanced by reducing NP size. Ultimately, in vivo studies have commonly shown that the transfection of nucleic acids into the lungs of mice is enhanced by PEGylation because of the improvement in mucus migration, irrespective of the fact that it typically shields the cationic surface charge that is required to facilitate cell binding [133]. The molecular weight of the attached PEG and its coating density on polymeric NP do, however, affect mucus penetration capability [134,135]. It must be acknowledged, though, that while PEGylation has often been shown to enhance the mucus penetration and lung cell transfection of polymer-based nucleic acid delivery systems, others have shown a lack of improvement over unmodified systems [136,137]. For this reason, the Loxegen technology employs a Zn-modified PEG that associates with the CPP-based gene delivery system via non-covalent interactions [138]. Thus, the system is designed to show initial enhanced mucus penetration via the PEG coating, which is then expected to be liberated to reveal the surface cationic charge and facilitate binding to anionic cell surface charges. The NP are then internalized via endocytosis into cells, where they release their DNA cargo into the cytoplasm, aided by the CPP carrier [128].

One issue with PEGylation is its widespread use and presence of anti-PEG antibodies in a substantial proportion of the population that could potentially affect the pharmacokinetic behavior of PEGylated nucleic acid delivery systems or their safety. However, other mucus penetrating moieties have been explored, such as chondroitin sulfate A (CS-A) and mannitol, that have demonstrated better mucus diffusion than unmodified NP. It is predicted that CS-A lowers the NP zeta potential and diminishes interaction with negatively charged mucins, while mannitol interrupts the mucin-mucin association or reduces the viscosity of mucus by enhancing water adsorption [139]. In another study, chitosan-based NP coated with poly(N-vinyl pyrrolidone) exhibited better penetration in sheep nasal mucosa than PEGylated NP [140]. None of these systems, however, have been examined in clinical trials and therefore their clinical utility can only be speculated.

3. Gene therapy vs small molecule CFTR modulators

As described earlier, the introduction of CFTR modulators to restore CFTR function has had a significant impact on the treatment of pwCF. While the main clinical benefits from CFTR modulators are improvements in lung function, improvements in overall health and systemic benefits, including weight gain and improved fertility, have been reported. It must be acknowledged, however, that while inhaled nucleic acid therapy has potential to improve lung function, limit systemic adverse effects compared to modulators and provide a treatment option for pwCF who are refractory to modulators, clinical benefit would be limited to improvements in lung function alone. Conversely, lung-related adverse effects also need to be carefully considered and monitored for inhaled nucleic acid therapies since these have the potential to negate any therapy benefit. Overall, a successful inhaled nucleic acid therapy would be expected to improve lung function by significantly increasing FEV1 and reduce pulmonary exacerbations while being well tolerated long term.

An important limitation of CFTR modulators is their high cost and inability to improve CFTR function in approximately 10% of patients with particular mutations, such as those with class I mutations, where no CFTR protein is produced. For these patients, inhaled gene and RNA therapy offers a clear solution to improving the management of the disease and alleviating the daily burden of intense physical and drug therapy, provided the new therapy can be made cost-effective and accessible by the broad pwCF population. We must consider, however, the advantages and disadvantages of oral modulator therapy versus inhaled gene or RNA therapy from the patient’s perspective. In doing this, we must consider the top 10 ‘questions for clinical research in CF’ determined by the James Lind Alliance Priority Setting Partnership in CF and published in 2023 [141]. Aside from priorities focused on lung infections and antibiotic treatment, key priorities included understanding the long-term positive and adverse effects of CFTR modulators, determining what options are available for people who cannot take modulators (including gene and RNA therapy), identifying effective ways of simplifying the treatment burden and preventing or alleviating CF-related diabetes and gastrointestinal symptoms.

With this in mind, the most effective CFTR modulator therapy is a twice-daily orally administered triple-drug combination comprised of elexacaftor, tezacaftor and ivacaftor (ETI, TRIKAFTA). Recently, however, a single daily triple-drug therapy was recently released containing vanzacaftor, tezacaftor and deutivacaftor (ALYFTREK) and is proposed to exhibit an improved ability to enhance CFTR function over TRIKAFTA. An advantage of this therapy is the simplicity of once or twice daily oral administration. A systematic review of this drug combination, involving publications from 2012 to September 2022 (nine clinical trials and 16 observational studies), reported an increase in lung function and other health parameters, including BMI, FEV₁, lung clearance index (LCI) and pulmonary exacerbations or sweat chloride concentration. Furthermore, this drug combination demonstrated a generally favorable safety profile [135] and benefited patients with advanced lung disease [142]. In addition to improvements in lung function and overall health, CFTR modulators have also been reported to improve pancreatic function and presumably delay or prevent the onset of CF-related diabetes [32], gastrointestinal and hepatobiliary effects, enhance fertility [143] and possibly, though currently under debate, immunity [144,145].

Despite improving the overall wellbeing of pwCF, CFTR modulators have limitations, including the inability to stop the decline in lung function. Disease progression (including lung infections, inflammation, pulmonary exacerbations and structural damage) continues, albeit at a slower rate compared to patients who do not take modulators. For example, a study of 161 pwCF given ivacaftor alone showed that reductions in the frequency of pulmonary exacerbations did not enhance the rate of total lung function recovery compared to placebo [140]. In addition, structural changes in the lung, such as alveolar simplification, bronchiectasis and cysts, cannot be reversed, and the susceptibility to lung infections remains [146].

The most significant downside to modulator therapy is the adverse side effect profile. The most common adverse effects include rash, headache, stomach pain, liver injury, and neuropsychiatric episodes, some of which are severe enough to justify medication withdrawal or prompt discontinuation by patients [147,148]. Depression-related adverse events after treatment with ETI have also been reported, despite some debate about the correlation between depression and this medication [149]. For instance, a safety data review from 24 clinical trials, CF databases in Germany and the United States, and post-marketing surveillance from 61,499 pwCF undergoing ETI treatment suggested that depression symptoms and depression-related effects do not have a causal relationship with the treatment but are related to the epidemiological background of the patients [150]. In contrast, a case review by Nidegger et al. (2025) revealed a correlation between ETI use and increased reporting of suicidal behavior [146]. Further, ETI has been reported to show adverse drug interactions with some drugs, such as several used in organ transplant recipients, as a result of competition for cytochrome P450 enzymes [151–153]. This is likely to become more problematic as the life expectancy of CF patients increases, since polypharmacy is common in the elderly.

Since modulator therapy is life-long, patients must weigh up the beneficial effects of modulator therapy with the potential for ongoing adverse effects, which can have a negative impact on wellbeing, as well as the high cost of these treatments to healthcare providers or patients not covered by healthcare. Nevertheless, abruptly stopping CFTR modulator therapy may cause disease rebound and severe lung exacerbations [154].

CFTR nucleic acid therapy has the clear potential to return CFTR function in those who are not eligible for, or who cannot tolerate, CFTR modulators. It is unknown at this stage what impact they would have on halting the progression of the disease in the lungs. While the transfection/transduction of 10–15% of epithelial cells may be sufficient to alleviate CF respiratory symptoms, it is not known whether this would be sufficient to prevent respiratory exacerbations, infections and structural aberrations. Further, in contrast to the positive effects of modulators on systemic disease, a major limitation of inhalable CFTR nucleic acid therapy is its inability to correct systemic CFTR function and have an impact on gastrointestinal symptoms and diabetes onset. The concept of a CF nucleic acid therapy administered systemically to target multiple organs is feasible, since most of the currently approved gene therapeutics are administered intravenously [155,156]. However, the focus of CF gene and RNA therapy to date has been on inhaled administration to specifically treat respiratory disease. Extrapolation to investigate systemically administered nucleic acid therapy could set the field back by at least a decade, particularly since most of the significant adverse effects associated with gene therapies relate to systemic administration, such as hepatotoxicity (which is also evident in modulator therapy) and thrombotic microangiopathy [156]. Further, systemic administration would require an exceptionally high dose to achieve sufficient distribution and transfection in key affected organs due to the propensity for liver distribution, which would significantly elevate the cost of CF gene therapy and make it inaccessible to much of the CF community.

The potential risk of genotoxicity and induction of cancers remains, particularly with genome-integrating viral gene delivery systems. However, with the lung-specific administration of nucleic acid therapies and the expected limited escape of these systems from the lungs, the risk of systemic cancers and other adverse effects is low. While the risk of lung-related cancers is possible, early detection and treatment as part of the routine close examination of the lungs of pwCF would reduce their impact. The bigger issue remains the significant potential for these therapies to exacerbate lung inflammation, which could temporarily worsen lung function. This may dictate the need for concomitant inhaled steroid treatment to reduce lung damage due to inflammation, along with the inhaled delivery of gene or RNA therapeutics, at the risk of increasing the frequency of lung infections. To date, inhaled CFTR gene and RNA therapies are reported to be generally well tolerated in patients, with relatively mild respiratory adverse effects. Other reported adverse effects are considered unrelated to the therapy itself. Of note, clinical trials undertaken so far have been relatively short-term, so it is still unknown what the long-term effects, if any, are likely to be. Additionally, repeated dose administration will be required for gene therapy since targeted epithelial cells are typically differentiated with a finite life-span [155,156]. However, nebulized administration of a nucleic acid therapy once every few weeks or less is relatively noninvasive, not overly time-consuming and would theoretically have similar or better effects on the lungs compared to the modulators. It therefore offers a benefit in terms of simplifying CF management and reducing treatment burden on patients, especially if the need for daily chest physiotherapy was no longer needed.

4. Conclusion

The promising results of inhaled CF gene and RNA therapy at the pre-clinical stage have failed to guarantee success in human clinical trials for over thirty years. This has raised questions in the minds of many in the field about the prospects of this therapeutic option. Despite the clinical use of many injectable gene therapies, penetration through the pulmonary mucus barrier, sufficient uptake into relevant airway cells and endosomal escape have been major barriers to returning CFTR function to the lungs after inhaled delivery. Knowledge gained from prior failures, particularly for viral delivery systems, have led to the development and current clinical testing of strategically designed and more advanced inhaled delivery systems that show early potential to make it past the Phase II hurdle. At present, the inhalable nucleic acid therapy landscape for CF is changing rapidly, with clinical failures being matched by early successes, particularly for viral vectors. Krystal Bio’s HSV-1-based vector is showing early promise in the treatment of CF, while the vector is slated to be the first clinically approved inhalable gene therapy for lung cancer. While inhalable liposome-based drug delivery systems are clinically approved, lipid-based carriers have, to date, shown mixed potential as inhalable gene and RNA therapies for CF. Polymeric delivery systems may present a viable option, but are still at the development and preclinical stage. While under characterized as an inhalable gene delivery system, they show properties that may overcome some of the limitations or concerns with viral and lipid-based delivery systems. However, more work is needed before these systems are ready to be benchmarked in clinical trials against viral and lipid-based competitors.

5. Future perspectives

The development of any nucleic acid therapy is a long journey, as shown by the first FDA-approved gene therapy (tisagenlecleucel), 30 years after the first publication of the concept [157]. The challenges in pulmonary delivery and the complexity of CF pathology might increase the waiting time until this therapeutic option is available in the market. Approval of inhalable gene or RNA therapies for CF may still be at least a decade away from becoming a reality, especially since the inhaled delivery of nanomaterials appears to be scrutinized more closely by regulators compared to injectable nanomedicines. That said, the FDA fast track approval for KB707 is a promising sign that inhalable nucleic acid therapies, and their approval, remain a real clinical possibility [158]. Even after approval, however, CFTR modulators are still likely to be recommended for ongoing management of CF since they have systemic effects, while inhalable gene therapies, though more targeted than the modulators, can only alleviate the respiratory aspects of the disease. The cost of treatment is also something that is likely to be a limiting factor for CFTR gene and RNA therapies. For this reason, the long-term success of inhalable CF gene and RNA therapies will be contingent upon the demonstration of superior return of pulmonary function when compared to the modulators.

Article highlights

• This review provides an objective overview of the progress and future of the inhaled delivery technologies that have been examined to date and the observed challenges and benefits of each. Recent advancements aimed at overcoming such challenges are presented. The benefits and downfalls of modulator therapy are compared to inhaled gene and RNA therapy with a view to evaluating how gene and RNA therapy will likely be positioned in the CF treatment space if this technology receives market approval.

• Sufficient CFTR RNA and gene delivery into the key airway cells of the lungs has been a major barrier to improving lung function in humans

• DNA Integrating viral delivery systems present risks of carcinogenesis that have been addressed by the use of non-integrating viruses and the strategic modification of integrating viruses. Other significant health risks commonly associated with clinically used systemically administered viral delivery systems are less likely to be an issue after inhaled delivery due to the typically poor penetration of nanomaterials through the lungs.

• Lipid-based delivery vectors are the only non-viral delivery systems that have been explored clinically for inhaled nucleic acid therapy for CF. They show good short-term safety in the lungs but in most cases, show a lack of significant efficacy compared to viral delivery systems

• Polymer-based delivery vectors have some advantages over viral and lipid-based delivery systems, but they have not yet been explored in clinical trials

• Consideration needs to be given to the potential cost of an inhalable RNA or gene therapy for CF if approved to ensure they are widely accessible. Notably, CFTR modulators come with a significant cost, making them an unfeasible treatment option for a large proportion of CF sufferers worldwide.

• Given the lack of significant improvement in respiratory function for inhaled CFTR RNA and gene delivery systems over the past 30 years, the future of this therapy is contingent upon the demonstration of clinical benefit for patients and long-term tolerability in current clinical trials.

• If an inhalable RNA or gene therapy is clinically approved for CF, CFTR modulators are still likely to be used concomitantly with the inhaled therapy to maximize clinical benefit in the lungs and improve CFTR function and clinical benefit systemically.

Supplementary Information

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17435889.2026.2640157