Molecular Therapy, 16 S309
The American Society of Gene Therapy Annual Conference, Boston, 2008
Obtaining evidence of successful gene transfer in human clinical samples is dependent on the sensitivity and robustness of the chosen clinical assay.
In preparation for Cystic Fibrosis (CF) gene therapy clinical trials, we have investigated a range of assays to evaluate the efficiency of gene transfer, including transgene-specific mRNA quantification by TaqMan RT-PCR.
Lipid/pDNA complexes consisting of GL67A and a novel CpG-free plasmid (pGM169) expressing the CF gene product (CFTR) will be nebulised to the lungs of CF patients. During bronchoscopy, airway epithelial cells will be collected to quantify plasmid-specific, human CFTR expression as a percentage of endogenous CFTR using quantitative RT-PCR. The requisite TaqMan assay must be highly sensitive in order to detect very small quantities of plasmid-specific RNA, whilst maintaining discrimination between RNA and large quantities of plasmid DNA delivered to the lung. RACE was used to confirm the sequence of the spliced mRNA in human cell culture, and RNA standards were produced to match this sequence. Then a range of TaqMan assays from >50 different combinations were evaluated. Assay RPS-169-B stood out in its sensitivity on RNA standards, detecting as few as 25-125 copies per RT PCR reaction. The design of the clinical plasmid, with a very short first exon (30bp), short intron (130bp), and a high GC content in the 5 UTR despite its CpG-free status, forced the forward primer, rather than the reverse primer, to span the exon junction, which implied the risk of the assay detecting DNA as well as RNA, and extensive DNase treatment of samples was required to guarantee an RNA-specific signal. The use of random hexamers, as opposed to a gene-specific primer for RT-PCR, resulted in no further improvement in sensitivity, however a nested PCR approach (assay RPS-169-F), with a RT-PCR-specific primer binding 3 of the TaqMan PCR reverse primer improved the assay sensitivity 10-fold, resulting in the detection of as few as 2.5 copies per RT-PCR reaction.
To confirm this in vivo, the clinical formulation GL67A/pGM169 was aerosolised to the lungs of mice, and the RPS-169-F assay permitted detection of pGM169 mRNA in 25/27 RNA samples, compared with 11/27 samples with RPS-169-B. The RNA/DNA discrimination of the new assay (RPS-169-F) was verified using similar mouse lung samples and required a double-DNase treatment. The optimisation of this quantitative RT-PCR clinical assay should allow us to detect small numbers of copies of plasmid-specific mRNA in human airway epithelial cells, which is crucial for the evaluation of gene transfer in human studies.
In addition our work demonstrates that one should bear in mind the constraints of TaqMan assay design during clinical plasmid development