Calculating the percentage of cells transfected following non-viral delivery to the respiratory epithelium. (2009)

Pringle, I. A., Hewitt, A.-M., Conolly, M. M., Lawton, A. E., Davies, J. C., Voase, N., Larsen, M., Manvelle, M., Donovan, J., Mohamedhossen, M. H., Alton, E. W. F. W., McLachlan, G., Collie, D. D. S., Hyde, S. C. & Gill, D. R.

Molecular Therapy, 17, S227

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Lung gene therapy has attracted both viral and non-viral vector candidates, but direct comparisons of vector performance in vivo are complicated by a variety of issues arising out of differences in animal models, delivery methods, antibody performance and reporter gene assay sensitivities. Only a robust, highly sensitive assay to quantify the number of transfected cells in the lung epithelium will permit a timely comparison of vector efficiency in animal models and ultimately in early clinical trials. Whereas quantitative RT-PCR is highly sensitive and can be used to measure expression levels of therapeutic genes in target cells (rather than compromise reporter genes), such assays tend to be solution-based and can misleadingly report high levels of mRNA from a small number of transfected cells; they cannot report the percentage of positive cells (PPC) in an epithelium.

To address this question, we have used rapid processing of samples (Cells-to-Ct, Ambion) for RT-PCR, and combined it with highly sensitive quantitative TaqMan RT-PCR, to develop a high-throughput assay that allows us to quantify the percentage positive cells (PPC) in a respiratory epithelium. The assay relies on the ability of RT-PCR to report the therapeutic gene expression in a sample of just 10 epithelial cells; repeating this 160 times for a given sample generates an estimate of PPC in that sample within the range of 0.06% - 10%. We aim to use the PPC assay on samples from our current clinical trial to assess the safety of aerosol delivery of pGM169/GL67A to the lungs of patients with Cystic Fibrosis (CF).

When HEK293T cells were transfected with clinical plasmid pGM169, detection of vector mRNA using Cells-to-Ct processing was 100-fold higher than using standard methods and confirmed positive detection of pGM169 mRNA from single cells. Detection of human endogenous CFTR mRNA from T84 cells, human Air Liquid Interface cultures and human respiratory epithelial cells collected from the nasal epithelium was also improved using Cells-to-Ct processing. Subsequently we confirmed that nasal brushing samples collected from human volunteers could be stored long-term (>2 months) in RNALater (Ambion) at 4oC. The cells were diluted to 1 cell/µl and 160 x 10 µl Cells-to-Ct samples created from each nasal brushing. Following analysis via Taqman RT-PCR, >95% of the samples were positive for human GAPDH mRNA. An important feature of the PPC assay is that the total number of cells required to perform the assay is minimal compared with traditional quantification of gene expression, and by storing the cells in RNALater the handling of samples by clinical staff is facile. In addition to using the PPC assay to estimate the percentage of nasal respiratory epithelial cells expressing vector mRNA in CF patient samples, the assay will be used to assess reliability of various human and animal lung models to predict clinical outcomes.

This type of technology could be further utilised to compare the relative transfection efficiency of viral and non-viral vectors in other organs.

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