An immunocytochemical assay to detect human CFTR expression following gene transfer.

Medical Genetics, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH42XU, UK.
Molecular and Cellular Probes (Impact Factor: 1.87). 08/2009; 23(6):272-80. DOI: 10.1016/j.mcp.2009.07.001
Source: PubMed

ABSTRACT To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery.
We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR.
The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells.
We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.

1 Bookmark
  • [Show abstract] [Hide abstract]
    ABSTRACT: The objective of an input design technique is to design an input to the system that results in an optimal tracking performance given some knowledge of the system response. Many such techniques focus on eliminating excitation of certain dominant system poles from the trajectory thus reducing any vibrations caused by these system poles. A downside to these methods is that they result in an elongation of the original trajectory. For a point-to-point control setting this means that there will be a trade-off between the elongation of the trajectory and the reduction of the settling time. In this paper will be shown that iterative learning control (ILC) can be used to design the input signal (trajectory) for a point-to-point motion in a way that eliminates all vibrations in the system without any elongation of the trajectory. This result is exactly the objective of classic input shaping techniques. The technique is illustrated with an application to a high precision wafer-stage.
    American Control Conference, 2003. Proceedings of the 2003; 07/2003
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.
    Biomaterials 09/2013; · 8.31 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding where mutant CFTR is localised in airway epithelia is essential in guiding the best therapeutic approach to correct the dysfunction of the CFTR protein. The widely held paradigm is that CF patients harbouring the commonest mutation, CFTR-delF508, trap CFTR within the endoplasmic reticulum and target it for degradation. However there are conflicting reports concerning expression and localisation of CFTR-delF508 in lung tissue. To attempt to resolve this fundamental issue we developed a novel approach to measure CFTR-delF508 in the lower airways of patients who have undergone lung transplantation for advanced CF. By sampling CF and non-CF epithelium simultaneously from the same individual, confounding factors of different airway microenvironments which may have influenced previous observations can be overcome. Epithelia sampled by bronchial brushing above (CF) and below (non-CF) the bronchial anastomosis were stained for CFTR and the localisation and level of expression assessed (n = 12). There was no significant difference in the proportion of tall columnar cells showing CFTR immunostaining as a discrete band at the apical membrane in cells harbouring the CFTR-delF508 mutation compared to non-CF cells (p = 0.21, n = 12). However, the amount of CFTR expressed at the apical surface was reduced by ∼50% in CF cells compared to non-CF cells (p = 0.04, n = 5). Our novel observation challenges the prevailing paradigm that CFTR is essentially absent from the apical membrane of respiratory cells harbouring the CFTR-delF508 mutation. Moreover, it raises the possibility that the new generation of CFTR potentiators may offer a realistic therapeutic option for CF patients.
    PLoS ONE 01/2011; 6(8):e23226. · 3.53 Impact Factor

Full-text (2 Sources)

Available from
May 21, 2014