Lentiviral vector gene therapy and CFTR modulators show comparable effectiveness in cystic fibrosis rat airway models.

Mutation-agnostic treatments such as airway gene therapy have the potential to treat any individual with cystic fibrosis (CF), irrespective of their CF transmembrane conductance regulator (CFTR) gene variants. The aim of this study was to employ two CF rat models, Phe508del and CFTR knockout (KO), to assess the comparative effectiveness of CFTR modulators and lentiviral (LV) vector-mediated gene therapy. Cells were isolated from the tracheas of rats and used to establish air-liquid interface (ALI) cultures. Phe508del rat ALIs were treated with the modulator combination, elexacaftor-tezacaftor-ivacaftor (ETI), and separate groups of Phe508del and KO tracheal epithelial cells were treated with LV-CFTR followed by differentiation at ALI. Ussing chamber measurements were performed to assess CFTR function. ETI-treated Phe508del ALI cultures demonstrated CFTR function that was 59% of wild-type level, while gene-addition therapy restored Phe508del to 68% and KO to 47% of wild-type level, respectively. Our findings show that rat Phe508del-CFTR protein can be successfully rescued with ETI treatment, and that CFTR gene-addition therapy provides significant CFTR correction in Phe508del and KO ALI cultures to levels that were comparable to ETI. These findings highlight the potential of an LV vector-based gene therapy for the treatment of CF lung disease.

Effective viral-mediated lung gene therapy: is airway surface preparation necessary?

Gene-based therapeutics are actively being pursued for the treatment of lung diseases. While promising advances have been made over the last decades, the absence of clinically available lung-directed genetic therapies highlights the difficulties associated with this effort. Largely, progress has been hindered by the presence of inherent physical and physiological airway barriers that significantly reduce the efficacy of gene transfer. These barriers include surface mucus, mucociliary action, cell-to-cell tight junctions, and the basolateral cell membrane location of viral receptors for many commonly used gene vectors. Accordingly, airway surface preparation methods have been developed to disrupt these barriers, creating a more conducive environment for gene uptake into the target airway cells. The two major approaches have been chemical and physical methods. Both have proven effective for increasing viral-mediated gene transfer pre-clinically, although with variable effect depending on the specific strategy employed. While such methods have been explored extensively in experimental settings, they have not been used clinically. This review covers the airway surface preparation strategies reported in the literature, the advantages and disadvantages of each method, as well as a discussion about applying this concept in the clinic.

Repeat or single-dose lentiviral vector administration to mouse lungs? It's all about the timing.

Lentiviral vectors are attractive delivery vehicles for cystic fibrosis gene therapy owing to their low immunogenicity and ability to integrate into the host cell genome, thereby producing long-term, stable gene expression. Nonetheless, repeat dosing may be required to increase initial expression levels, and/or boost levels when they wane. The primary aim of this study was to determine if repeat dosing of a VSV-G pseudotyped LV vector delivered into mouse lungs is more effective than a single dose. C57Bl/6 mouse lungs were conditioned with lysophosphatidylcholine, followed one-hour later by a LV vector carrying the luciferase reporter gene, using six different short-term (≤1 wk) and long-term (>1 wk) dosing schedules. Luciferase expression was quantified using bioluminescence imaging over 12 months. Most dosing schedules produced detectable bioluminescence over the 12-month period, but the shorter intervals (≤1 wk) produced higher levels of flux than the longest interval (five doses at least 1-month apart). Ex vivo lung analysis at 12 months showed that the estimated mean flux for the group that received two doses 1-week apart was significantly greater than the single dose group and the two groups that received doses over a period greater than 1-week. These results suggest that early consecutive multiple doses are more effective at improving gene expression in mouse lungs at 12 months, than longer repeat dosing intervals.

Changes in Essential Fatty Acids and Ileal Genes Associated with Metabolizing Enzymes and Fatty Acid Transporters in Rodent Models of Cystic Fibrosis.

Cystic fibrosis (CF), the result of mutations in the CF transmembrane conductance regulator (CFTR), causes essential fatty acid deficiency. The aim of this study was to characterize fatty acid handling in two rodent models of CF; one strain which harbors the loss of phenylalanine at position 508 (Phe508del) in CFTR and the other lacks functional CFTR (510X). Fatty acid concentrations were determined using gas chromatography in serum from Phe508del and 510X rats. The relative expression of genes responsible for fatty acid transport and metabolism were quantified using real-time PCR. Ileal tissue morphology was assessed histologically. There was an age-dependent decrease in eicosapentaenoic acid and the linoleic acid:α-linolenic acid ratio, a genotype-dependent decrease in docosapentaenoic acid (n-3) and an increase in the arachidonic acid:docosahexaenoic acid ratio in Phe508del rat serum, which was not observed in 510X rats. In the ileum, Cftr mRNA was increased in Phe508del rats but decreased in 510X rats. Further, Elvol2, Slc27a1, Slc27a2 and Got2 mRNA were increased in Phe508del rats only. As assessed by Sirius Red staining, collagen was increased in Phe508del and 510X ileum. Thus, CF rat models exhibit alterations in the concentration of circulating fatty acids, which may be due to altered transport and metabolism, in addition to fibrosis and microscopic structural changes in the ileum.

Animal and Cell Culture Models for Cystic Fibrosis: Which Model Is Right for Your Application?

Over the past 30 years, a range of cystic fibrosis (CF) animal models have been generated for research purposes. Different species, including mice, rats, ferrets, rabbits, pigs, sheep, zebrafish, and fruit flies, have all been used to model CF disease. While access to such a variety of animal models is a luxury for any research field, it also complicates the decision-making process when it comes to selecting the right model for an investigation. The purpose of this review is to provide a guide for selecting the most appropriate CF animal model for any given application. In this review, the characteristics and phenotypes of each animal model are described, along with a discussion of the key considerations that must be taken into account when choosing a suitable animal model. Available in vitro systems of CF are also described and can offer a useful alternative to using animal models. Finally, the future of CF animal model generation and its use in research are speculated upon.

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing.

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

Methods for dynamic synchrotron X-ray respiratory imaging in live animals.

Small-animal physiology studies are typically complicated, but the level of complexity is greatly increased when performing live-animal X-ray imaging studies at synchrotron and compact light sources. This group has extensive experience in these types of studies at the SPring-8 and Australian synchrotrons, as well as the Munich Compact Light Source. These experimental settings produce unique challenges. Experiments are always performed in an isolated radiation enclosure not specifically designed for live-animal imaging. This requires equipment adapted to physiological monitoring and test-substance delivery, as well as shuttering to reduce the radiation dose. Experiment designs must also take into account the fixed location, size and orientation of the X-ray beam. This article describes the techniques developed to overcome the challenges involved in respiratory X-ray imaging of live animals at synchrotrons, now enabling increasingly sophisticated imaging protocols.

Airway disease phenotypes in animal models of cystic fibrosis.

In humans, cystic fibrosis (CF) lung disease is characterised by chronic infection, inflammation, airway remodelling, and mucus obstruction. A lack of pulmonary manifestations in CF mouse models has hindered investigations of airway disease pathogenesis, as well as the development and testing of potential therapeutics. However, recently generated CF animal models including rat, ferret and pig models demonstrate a range of well characterised lung disease phenotypes with varying degrees of severity. This review discusses the airway phenotypes of currently available CF animal models and presents potential applications of each model in airway-related CF research.

Insights into epithelial-mesenchymal transition from cystic fibrosis rat models.

BACKGROUND: Molecular pathways contributing to Cystic Fibrosis pathogenesis remain poorly understood. Epithelial-mesenchymal transition (EMT) has been recently observed in CF lungs and certain CFTR mutation classes may be more susceptible than others. No investigations of EMT processes in CF animal models have been reported. AIM: The aim of this study was to assess the expression of EMT-related markers in Phe508del and knockout (CFTR-KO) rat lung tissue and tracheal-derived basal epithelial stem cells, to determine whether CFTR dysfunction can produce an EMT state. METHOD: The expression of EMT-related markers in lung tissue and cultured tracheal basal epithelial stem cells from wildtype (WT), Phe508del, and CFTR-KO rats were assessed using qPCR and Western blots. Cell responses were evaluated in the presence of Rho-associated protein kinase (ROCK) inhibitor Y27632, which blocks EMT-pathways, or after treatment with TGFß1 to stimulate EMT. RESULTS: Different gene expression profiles were observed between Phe508del and CFTR-KO rat models compared to wild type. There was lower expression of type 1 collagen in KO lungs and primary cell cultures, while Phe508del lungs and cells had higher expression, particularly when treated with TGFß1. The addition of Y27632 rescued changes in EMT related genes in Phe508del cells but not in KO cells. CONCLUSION: Our findings show the first evidence of upregulated EMT pathways in the lungs and airway cells of any CF animal model. Differences in the regulation of the EMT genes and proteins in the Phe508del and CFTR-KO cells suggest that the signalling pathways underlying EMT are CFTR mutation dependent.

Ultra-fastin vivodirectional dark-field x-ray imaging for visualising magnetic control of particles for airway gene delivery.

Objective.Magnetic nanoparticles can be used as a targeted delivery vehicle for genetic therapies. Understanding how they can be manipulated within the complex environment of live airways is key to their application to cystic fibrosis and other respiratory diseases.Approach.Dark-field x-ray imaging provides sensitivity to scattering information, and allows the presence of structures smaller than the detector pixel size to be detected. In this study, ultra-fast directional dark-field synchrotron x-ray imaging was utlilised to understand how magnetic nanoparticles move within a live, anaesthetised, rat airway under the influence of static and moving magnetic fields.Main results.Magnetic nanoparticles emerging from an indwelling tracheal cannula were detectable during delivery, with dark-field imaging increasing the signal-to-noise ratio of this event by 3.5 times compared to the x-ray transmission signal. Particle movement as well as particle retention was evident. Dynamic magnetic fields could manipulate the magnetic particlesin situ. Significance.This is the first evidence of the effectiveness ofin vivodark-field imaging operating at these spatial and temporal resolutions, used to detect magnetic nanoparticles. These findings provide the basis for further development toward the effective use of magnetic nanoparticles, and advance their potential as an effective delivery vehicle for genetic agents in the airways of live organisms.

Corrigendum: Effect of elexacaftor-tezacaftor-ivacaftor on nasal potential difference and lung function in Phe508del rats.

[This corrects the article DOI: 10.3389/fphar.2024.1362325.].

Effect of elexacaftor-tezacaftor-ivacaftor on nasal potential difference and lung function in Phe508del rats.

Introduction: Phe508del is the most common cystic fibrosis transmembrane conductance regulator (CFTR) gene variant that results in the recessive genetic disorder cystic fibrosis (CF). The recent development of highly effective CFTR modulator therapies has led to significant health improvements in individuals with this mutation. While numerous animal models of CF exist, few have a CFTR mutation that is amenable to the triple combination therapy elexacaftor-tezacaftor-ivacaftor (ETI). Methods: To determine the responsiveness of Phe508del rats to ETI, a baseline nasal potential difference was measured. Subsequently, they received ETI daily for 14 days, after which post-treatment nasal potential difference, lung mechanics (via flexiVent) and lung ventilation (via X-ray Velocimetry) were assessed. Results: Chloride ion transport in nasal airways was restored in Phe508del rats treated with ETI, but neither lung mechanics nor ventilation were significantly altered. Discussion: These findings validate the usefulness of this rat model for future investigations of modulator therapy in CF.

Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine.

The Ca2+ activated Cl- channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.

To bead or not to bead: A review of Pseudomonas aeruginosa lung infection models for cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterised by recurring bacterial infections resulting in inflammation, lung damage and ultimately respiratory failure. Pseudomonas aeruginosa is considered one of the most important lung pathogens in those with cystic fibrosis. While multiple cystic fibrosis animal models have been developed, many fail to mirror the cystic fibrosis lung disease of humans, including the colonisation by opportunistic environmental pathogens. Delivering bacteria to the lungs of animals in different forms is a way to model cystic fibrosis bacterial lung infections and disease. This review presents an overview of previous models, and factors to consider when generating a new P. aeruginosa lung infection model. The future development and application of lung infection models that more accurately reflect human cystic fibrosis lung disease has the potential to assist in understanding the pathophysiology of cystic fibrosis lung disease and for developing treatments.

Essential Fatty Acid Deficiency in Cystic Fibrosis Disease Progression: Role of Genotype and Sex.

Adequate intake of nutrients such as essential fatty acids (EFA) are critical in cystic fibrosis (CF). The clinical course of deterioration of lung function in people with CF has been shown to relate to nutrition. Independent of the higher energy consumption and malabsorption due to pancreatic insufficiency, EFA deficiency is closely associated with the risk of pulmonary infection, the most significant pathology in CF. This review will focus on the EFA deficiency identified in people with CF, as well as the limited progress made in deciphering the exact metabolic pathways that are dysfunctional in CF. Specifically, people with CF are deficient in linoleic acid, an omega 6 fatty acid, and the ratio of arachidonic acid (omega 6 metabolite) and docosahexaenoic acid (omega 3 metabolite) is increased. Analysis of the molecular pathways in bronchial cells has identified changes in the enzymes that metabolise EFA. However, fatty acid metabolism primarily occurs in the liver, with EFA metabolism in CF liver not yet investigated, indicating that further research is required. Despite limited understanding in this area, it is well known that adequate EFA concentrations are critical to normal membrane structure and function, and thus are important to consider in disease processes. Novel insights into the relationship between CF genotype and EFA phenotype will be discussed, in addition to sex differences in EFA concentrations in people with CF. Collectively, investigating the specific effects of genotype and sex on fatty acid metabolism may provide support for the management of people with CF via personalised genotype- and sex-specific nutritional therapies.

Comparison of Physical Perturbation Devices for Enhancing Lentiviral Vector-Mediated Gene Transfer to the Airway Epithelium.

Natural airway defenses currently impede the efficacy of viral vector-mediated airway gene therapy. Conditioning airways before vector delivery can disrupt these barriers, improving viral vector access to target receptors and airway stem cells. This study aimed to assess and quantify the in vivo histological and gene transfer effects of physical perturbation devices to identify effective conditioning approaches. A range of flexible wire baskets with varying configurations, a Brush, biopsy forceps, and a balloon catheter were examined. We first evaluated the histological effects of physical perturbation devices in rat tracheas that were excised 10 min after conditioning. Based on the histological findings, a selection of devices was used to condition rat tracheas in vivo before delivering a lentiviral vector containing the LacZ reporter gene. After 7 days, excised tracheas were X-gal processed and examined en face to quantify the area of LacZ staining. Histological observations 10 min after conditioning found that physical perturbation dislodged cells from the basement membrane to varying degrees, with some producing significant levels of epithelial cell removal. When a subset of devices was assessed for their ability to enhance gene transfer, only the NGage® wire basket (Cook Medical) produced a significant increase in the proportion of X-gal-stained area when compared with unconditioned tracheas (eightfold, p = 0.00025). These results suggest that a range of factors contribute to perturbation-enhanced gene transfer. Overall, this study supports existing evidence that physical perturbation can assist airway gene transfer and will help to identify the characteristics of an effective device for airway gene therapy.

Improved in-vivo airway gene transfer via magnetic-guidance, with protocol development informed by synchrotron imaging.

Gene vectors to treat cystic fibrosis lung disease should be targeted to the conducting airways, as peripheral lung transduction does not offer therapeutic benefit. Viral transduction efficiency is directly related to the vector residence time. However, delivered fluids such as gene vectors naturally spread to the alveoli during inspiration, and therapeutic particles of any form are rapidly cleared via mucociliary transit. Extending gene vector residence time within the conducting airways is important, but hard to achieve. Gene vector conjugated magnetic particles that can be guided to the conducting airway surfaces could improve regional targeting. Due to the challenges of in-vivo visualisation, the behaviour of such small magnetic particles on the airway surface in the presence of an applied magnetic field is poorly understood. The aim of this study was to use synchrotron imaging to visualise the in-vivo motion of a range of magnetic particles in the trachea of anaesthetised rats to examine the dynamics and patterns of individual and bulk particle behaviour in-vivo. We also then assessed whether lentiviral-magnetic particle delivery in the presence of a magnetic field increases transduction efficiency in the rat trachea. Synchrotron X-ray imaging revealed the behaviour of magnetic particles in stationary and moving magnetic fields, both in-vitro and in-vivo. Particles could not easily be dragged along the live airway surface with the magnet, but during delivery deposition was focussed within the field of view where the magnetic field was the strongest. Transduction efficiency was also improved six-fold when the lentiviral-magnetic particles were delivered in the presence of a magnetic field. Together these results show that lentiviral-magnetic particles and magnetic fields may be a valuable approach for improving gene vector targeting and increasing transduction levels in the conducting airways in-vivo.

Single-Dose Lentiviral Mediated Gene Therapy Recovers CFTR Function in Cystic Fibrosis Knockout Rats.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective ion transport in the airways. Addition of a functioning CFTR gene into affected airway cells has the potential to be an effective treatment for lung disease. The therapeutic efficacy of airway gene transfer can be quantified in animal models by assessing ion transport in the treated nasal epithelium using the nasal potential difference (PD) measurement technique. The nasal PD technique is routinely used in CF mice, however when applied to a recently developed CF rat model those animals did not tolerate the initial nasal PD assessment, therefore the procedure was firstly optimised in rats. This study evaluated the effect of lentiviral (LV)-mediated CFTR airway gene delivery on nasal PD in a CFTR knockout rat model. LV gene vector containing the CFTR gene tagged with a V5 epitope tag (LV-V5-CFTR) was delivered to the nasal epithelium of CF rats, and one week later nasal PD was analysed. This study demonstrated for the first time that LV-V5-CFTR treatment produced a mean correction of 46% towards wild-type chloride response in treated CF rats. Transduced cells were subsequently identifiable using V5 immunohistochemical staining. These findings in the nose validate the use of airway gene therapy for future lung based experiments.

Breaching the Delivery Barrier: Chemical and Physical Airway Epithelium Disruption Strategies for Enhancing Lentiviral-Mediated Gene Therapy.

The lungs have evolved complex physical, biological and immunological defences to prevent foreign material from entering the airway epithelial cells. These mechanisms can also affect both viral and non-viral gene transfer agents, and significantly diminish the effectiveness of airway gene-addition therapies. One strategy to overcome the physical barrier properties of the airway is to transiently disturb the integrity of the epithelium prior to delivery of the gene transfer vector. In this study, chemical (lysophosphatidylcholine, LPC) and physical epithelium disruption using wire abrasion were compared for their ability to improve airway-based lentiviral (LV) vector mediated transduction and reporter gene expression in rats. When luciferase expression was assessed at 1-week post LV delivery, LPC airway conditioning significantly enhanced gene expression levels in rat lungs, while a long-term assessment in a separate cohort of rats at 12 months revealed that LPC conditioning did not improve gene expression longevity. In rats receiving physical perturbation to the trachea prior to gene delivery, significantly higher LacZ gene expression levels were found when compared to LPC-conditioned or LV-only control rats when evaluated 1-week post gene transfer. This proof-of-principle study has shown that airway epithelial disruption strategies based on physical perturbation substantially enhanced LV-mediated airway gene transfer in the trachea.

The Effects of Conditioning and Lentiviral Vector Pseudotype on Short- and Long-Term Airway Reporter Gene Expression in Mice.

A gene addition therapy into the conducting airway epithelium is a potential cure for cystic fibrosis lung disease. Achieving sustained lung gene expression has proven difficult due to the natural barriers of the lung. The development of lentiviral (LV) vectors pseudotyped with viral envelopes that have a natural tropism to the airway has enabled persistent gene expression to be achieved in vivo. The aims of this study were to compare the yields of hemagglutinin (HA) and vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped HIV-1 vectors produced under the same conditions by our standard LV vector production method. We then sought to measure gene expression in mouse airways and to determine whether lysophosphatidylcholine (LPC) conditioning enhances short- and long-term gene expression. C57Bl/6 mouse airways were conditioned with 10 µL of 0.1% LPC or saline control, followed 1 h later by a 30 µL dose of an HA or VSV-G pseudotyped vector carrying either the LacZ or luciferase reporter genes. LacZ expression was assessed by X-gal staining after 7 days, while lung luminescence was quantified regularly for up to 18 months by bioluminescent imaging. The HA pseudotyped vectors had functional titers 25 to 60 times lower than the VSV-G pseudotyped vectors. Conditioning the lung with LPC significantly increased the total number of LacZ-transduced cells for both pseudotypes compared to saline control. Regardless of LPC conditioning, the VSV-G pseudotype produced higher initial levels of gene expression compared to HA. LPC conditioning did not increase the number of transduced basal cells for either pseudotype compared to saline, and was not required for long-term gene expression. Both pseudotyped vectors effectively transduced the upper conducting airways of wild-type mice. The use of LPC conditioning before vector delivery was not required in mouse lungs to produce long-term gene expression, but did improve short-term gene expression.