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

Longevity of airway gene therapy for cystic fibrosis : single and repeat lentiviral dosing.

The promise of gene therapy as a treatment and/or cure for cystic fibrosis (CF) airway disease is yet to be fulfilled. Lentiviral (LV) vectors possess many of the properties that would satisfy the requirements for an effective clinical gene correction treatment; the capacity to hold the large CF transmembrane conductance regulator (CFTR) gene, pseudotyped envelopes that provide broad tropism for a range of cells and tissue types, the ability to transduce dividing and non-dividing cells, the potential for long-term gene expression from genomic integration, and the lack of pre-existing blocking antibodies for majority of the CF population. To determine the persistence of LV gene expression, the same mice were repeatedly assessed throughout their lifetimes. The utilization of the biological compound lysophosphatidylcholine (LPC) as a pre-treatment enhanced nasal airway gene expression of the HIV-1 based LV vector containing reporter genes, or the functional CFTR gene, in normal and CF mice in vivo. Nasal luciferase (Luc) gene expression from a single LPC/LV nasal dose was sustained for the life time of normal mice, possibly suggesting an involvement of stem/progenitor cells or long-lived terminally differentiated cells. In contrast, stable long-term Luc gene expression was detected in the lung airways without the requirement of LPC pre-treatment. The loss then re-emergence of lung luminescence in CF mice demonstrated that stem/progenitor cells were transduced. This was the first examination of persistence of LV reporter gene and functional gene expression, in individual CF mice over their lifetimes. CF mice treated with LPC/LV-CFTR demonstrated a significant partial functional correction of the nasal CFTR electrophysiological defect that was sustained for up to 1 year. Importantly, this significantly increased survival, close to that observed in normal mice. Since the level of functional expression diminished over time in CF mice the ability to re-dose and evade blocking host immune responses was addressed. Multiple doses of a LV vector over a short time frame were feasible but did not significantly increase expression compared to a single dose. Circulating antibodies to both the vector envelope and the transgene protein were detected after repeat dosing conducting over a longer time frame. The timing of additional LV vector doses may be crucial for effective boosting of waning gene expression. The addition of a transient immunosuppressive treatment did not significantly enhance the level of gene expression produced by a single dose, but did reduce circulating antibodies to both the delivered foreign transgene and to the pseudotyped envelope protein. The demonstration of longevity of gene expression, the functional correction of the CFTR defect, the substantial increase in CF animal survival, the ability to re-dose and the use of immune-suppression to reduce antibody production provides strong and specific support for the continued investigation of LV CFTR gene transfer towards a clinical gene therapy treatment for CF airway disease.Thesis (Ph.D.) -- -University of Adelaide, School of Paediatrics & Reproductive Health, 201

Epithelial disruption: a new paradigm enabling human airway stem cell transplantation

Abstract Background Airway disease is a primary cause of morbidity and early mortality for patients with cystic fibrosis (CF). Cell transplantation therapy has proven successful for treating immune disorders and may have the potential to correct the airway disease phenotype associated with CF. Since in vivo cell delivery into unconditioned mouse airways leads to inefficient engraftment, we hypothesised that disrupting the epithelial cell layer using the agent polidocanol (PDOC) would facilitate effective transplantation of cultured stem cells in mouse nasal airways. Methods In this study, 4 μL of 2% PDOC in phosphate-buffered saline was administered to the nasal airway of mice to disrupt the epithelium. At 2 or 24 h after PDOC treatment, two types of reporter gene-expressing cells were transplanted into the animals: luciferase-transduced human airway basal cells (hABC-Luc) or luciferase-transduced human amnion epithelial cells (hAEC-Luc). Bioluminescence imaging was used to assess the presence of transplanted luciferase-expressing cells over time. Data were evaluated by using two-way analysis of variance with Sidak’s multiple comparison. Results Successful transplantation was observed when hABCs were delivered 2 h after PDOC but was absent when transplantation was performed 24 h after PDOC, suggesting that a greater competitive advantage for the donor cells is present at the earlier time point. The lack of transplantation of hAECs 24 h after PDOC supports the importance of choosing the correct timing and cell type to facilitate transplantation. Conclusions These studies into factors that may enable successful airway transplantation of human stem cells showed that extended functioning cell presence is feasible and further supports the development of methods that alter normal epithelial layer integrity. With improvements in efficacy, manipulating the airway epithelium to make it permissive towards cell transplantation may provide another option for safe and effective correction of CF transmembrane conductance regulator function in CF airways

Directional Dark-Field Imaging Data of Magnet Assisted Airway Treatment Delivery

Image sequences taken using a single-grid imaging system at the BL20XU beamline of the SPring-8 synchrotron. Showing the delivery of magnetic nanoparticles into the trachea of a live rat for the purpose of developing a magnetically guided gene therapy delivery system.The images can be analysed with the UMPA Directional Dark-Field code available at: github.com/optimato/UMPA_directional_dark_field DOI for code is: doi.org/10.5281/zenodo.6981376 </p