Pediatric Pulmonology, 44 S32 Abstract 271
The North American Cystic Fibrosis Conference, Minneapolis, 2009
Gene therapy is being evaluated for treatment of chronic Cystic Fibrosis (CF) lung disease. When plasmid DNA expressing CFTR is complexed with cationic liposomes or polymers, it can be delivered to the lungs of mice and sheep via aerosol, and CFTR mRNA can be detected in lung tissue. Recently, the CpG-free plasmid pGM169, which can result in persistent, inflammation-free, expression of human CFTR via the hCEFI promoter in the mouse lung (Hyde et al 2008 Nature Biotechnology 26:549) was selected for clinical study. Currently, the UK Cystic Fibrosis Gene Therapy Consortium is conducting a Phase I clinical trial to evaluate the safety of aerosol delivery of a pGM169/GL67A liposome formulation to the lungs of CF patients. Although the primary aim of the clinical study is to evaluate safety, we are also taking the opportunity to measure gene expression in nasal and bronchial brushings. To quantify transgene expression in clinical samples, traditional 2-step TaqMan RT-PCR is routinely used as a reliable and sensitive method. Using this type of assay we are capable of quantifying pGM169 mRNA and endogenous human CFTR mRNA down to 25 and 10 copies/µl of input RNA respectively, however below this threshold the samples are non-quantifiable and are reported as negative. To improve the opportunity to detect low levels of mRNA in clinical samples we developed a 3-step TaqMan RT-PCR assay, which includes an additional nested PCR step. This resulted in improved sensitivity by facilitating detection of low concentration pGM169 mRNA mimics that were previously undetectable by 2-step TaqMan RT-PCR. To model expression of pGM169 in the human lung, sheep lung tissue samples were collected following aerosol delivery (32mg pGM169). When vector-specific CFTR mRNA was assayed in the sheep samples, 25% of samples reported as negative by 2-step TaqMan PCR, had a strong (though non-quantifiable) vector signal when assayed by 3-step TaqMan PCR. Similarly, RNA from human Air Liquid Interface cells transfected with pGM169 resulted in detection of vector signal in 50% of previously 'negative' samples. Thus application of the 3-step RT-PCR assay to RNA samples below the quantifiable threshold demonstrated that these tissue samples actually contained vector RNA signal. The ability to detect low levels of vector-specific mRNA in heterogeneous tissue samples indicates that this assay is ideal for measuring transgene expression in clinical samples, where availability may be limited.