Wave I: Current Clinical Trial

• Optimisation of plasmid DNA
• Aerosol delivery of gene transfer vectors
• Assay development

Wave II: The Future

• Viral vector development

Resources

• Core Facilities
• Patents

Assay Development

CFTR protein is shown at the apical surface of the cells (red signal).  Cytoplasm labelled with anti Cytokeratin antibody is  green and the nuclei are blue (DAPI). CFTR protein is shown at the apical surface of the cells (red signal).  Cytoplasm labelled with anti Cytokeratin antibody is  green and the nuclei are blue (DAPI).

In parallel with the development of gene therapy products there has been the development of clinical assays to gauge the effectiveness of gene therapy.

In many ways this type of research is as demanding as the development of the vectors themselves because without sufficiently powerful assays, it is impossible to determine the effectiveness of our technologies.

The types of assays we have developed can be broken down into a number of groups.

Clinical Biomarkers
Imaging Gene Transfer
Quantitative RT-PCR
Electrical Measurements of CFTR Function

Clinical Biomarkers^Top

Clinical biomarkers are assays that can be performed to give a reliable measurement of the degree of disease severity in a disease. In CF spirometry testing of lung function has been a classic biomarker for decades. However, for gene therapy clinical studies we wanted to be able to determine very subtle differences between patients that had received gene transfer vectors and those that had not. This required much more sensitive assays to be developed.

We have looked at a number of areas and you can find publications relating to these below. In particular we have been successful in developing lung clearance index assays (LCI) and believe this may be a more suitable approach to measuring changes in lung function in patients undergoing clinical trials.

Quantification of Periciliary Fluid (PCL) Height in Human Airway Biopsies is Feasible, but not Suitable as a Biomarker (2010). Griesenbach, U. et al., Am J Respir Cell Mol Biol, Advanced, 20418361.

Lung clearance index in CF: a sensitive marker of lung disease severity (2008). Davies, J. C. et al., Thorax, 63, 96-97.

Sputum proteomics in inflammatory and suppurative respiratory diseases (2008). Gray, R. D. et al., Am J Respir Crit Care Med, 178, 444-452.

Lung clearance index is a sensitive, repeatable and practical measure of airways disease in adults with cystic fibrosis (2008). Horsley, A. R. et al., Thorax, 63, 135-140.

Biomarkers for cystic fibrosis lung disease: application of SELDI-TOF mass spectrometry to BAL fluid (2008). MacGregor, G. et al., J Cyst Fibros, 7, 352-358.

Staining of clilia on an airway epithelium.

Imaging Gene Transfer^Top

One of the most meaningful ways of assessing the effectiveness of gene transfer efficiency is to use direct imaging technologies. For many preclinical studies we use fluorescent or luminescent reporter genes to be able to identify the cell types we have transfected in our models.

This allows us to answer one of the fundamental questions regarding gene transfer efficiency, "How many cells are we transfecting and how much do they express?"

Increasingly we are also using direct in-vivo imaging to make repeated measures of gene transfer in mice or in our ex-vivo models, thus reducing the number of animals and expense involved.

In many clinical trials, antibodies have been used to directly image CFTR protein expression. However, the range of antibodies available is limiting. Nevertheless our Antibody Working Group has developed a range of improved protocols to optimise such assays in our clinical trial and pre-clinical models.

Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer (2011). Griesenbach, U. et al., Biomaterials, 32, 2614-2624.

An immunocytochemical assay to detect human CFTR expression following gene transfer (2009). Davidson, H. et al., Mol Cell Probes, 23, 272-280.

In vivo imaging of gene transfer to the respiratory tract (2008). Griesenbach, U. et al., Biomaterials, 29, 1533-1540.

Identification of transfected cell types following non-viral gene transfer to the murine lung (2007). Davies, L. A. et al., J Gene Med, 9, 184-196.

Quantitative RT-PCR^Top

Imaging and biomarkers are very powerful tools to use to assess gene transfer. However, they are not truly quantitative and often fail to detect gene expression from inefficient gene transfer systems. This can be particularly problematic for early stage projects where maximal sensitivity is desirable.

Therefore the Consortium has a Taqman PCR core facility which is used to detect the presence of vector mRNA or DNA from most of our model systems and our ongoing clinical trial. The sensitivity of Taqman is far greater than most other assays available and can be used to detect <5 copies of mRNA or DNA in a sample.

Electrical Measurements of CFTR Function^Top

CFTR forms an ion channel and passage of ions through this channel can be measured electrically. In the clinical trial and in pre-clinical models we continue to make extensive use of such methods to assess the activity of the CFTR protein following gene transfer.

Validation of nasal potential difference measurements in gut-corrected CF knockout mice (2008). Griesenbach, U. et al., Am J Respir Cell Mol Biol, 39, 490-496.