Development of highly sensitive TaqMan RT-PCR assays for quantifying vector mRNA in human samples. (2008)

Sumner-Jones, S. G., Pringle, I. A., Davies, L. A., Coles, R. L., Painter, H., Evans, N., McCormick, D., Stoneham, S., Collie, D. D. S., McLachlan, G., Hyde, S. C. & Gill, D. R.

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In preparation for Cystic Fibrosis gene therapy clinical trials, we have investigated the use of transgene-specific mRNA quantification by TaqMan RT-PCR to obtain evidence of successful gene transfer. We tested lipid GL67A complexed with either the previously used clinical plasmid pCF1-CFTR, or a novel CpG-free plasmid pGM169 in mouse and sheep preclinical studies. Whole mouse lungs, or bronchial brushings and biopsies, similar to expected clinical samples, from treated sheep were analysed. The existing pCF1-CFTR assay typically allows detection of 2.5 copies per RT-PCR reaction. To obtain a pGM169 assay with sufficient sensitivity to detect small quantities of plasmid-specific RNA, whilst maintaining appropriate discrimination between RNA and large quantities of pDNA delivered to the lung, over 50 assays were tested. One assay (RPS-169-B) detected 25-125 copies per RT PCR reaction, but was further improved using a nested PCR approach with a RT-PCR-specific primer (RPS-169-F), resulting in routine detection of as few as 2.5 copies per reaction. To further confirm the improved sensitivity, the clinical formulation was aerosolised to the lungs of mice, and the RPS-169-F assay detected pGM169 mRNA in 25/27 RNA samples, compared with 11/27 samples using RPS-169-B. The design of pGM169 plasmid forced the forward primer to span the exon junction, which implied the risk of the assay detecting DNA as well as RNA. In contrast, it is the probe in the pCF1-CFTR primer set that spans the exon junction. This was reflected in the RNA/DNA discrimination of the two assays (RPS-pCF1-A and RPS-169-F): using mouse and sheep lung samples, we showed that pGM169 mRNA detection required a double-DNase treatment to guarantee an RNA-specific signal, whereas none was required for pCF1-CFTR. The optimisation of this quantitative RT-PCR assay should allow us to detect small numbers of copies of plasmid-specific mRNA in human airway epithelial cells following clinical delivery.

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