S94 Development of assays to assess safety and efficacy of lentiviral gene therapy for cystic fibrosis

Introduction The UK Cystic Fibrosis Gene Therapy Consortium has developed a novel lentiviral vector (rSIV.F/HN) designed to transduce airway epithelial cells efficiently and is preparing a first-in-man lentiviral gene therapy trial building on the recent success of the repeat-dosing non-viral trial. Assays to determine safety and efficacy of lentivirus-mediated gene transfer are now being evaluated. Here, we report validation of two comparatively novel techniques. Methods and Results Digital droplet (DD) RT-PCR: Virus particles may be shed over time following topical administration to the airways. DD-RT-PCR allows absolute and sensitive quantification of vector genomes. Saliva and urine samples were spiked with known quantities of vector particles (VP) to determine recovery and lower limit of detection (LLD) of the assay. Recovery rates in both body fluids were dependent on the amount of input RNA (Saliva: 7.1%–15.7% when spiked with 20–400 VP/uL, urine: 42.5%–76% when spiked with 20–400 VP/uL). The LLD is 200 and 400 VP/ml in urine and saliva, respectively. However, in an average batch only approximately 1:700 to 1:1000 VP is able to transduce a cell. In situ hybridisation Quantification of the number of airway cells transduced after pulmonary gene transfer has, to date, not been feasible. In situ hybridisation has traditionally suffered from poor sensitivity and high signal-to-noise ratio. RNAScope (ACDBio) is a novel in situ hybridisation technique based on overlapping probes and multiple levels of signal amplification to enhance specificity and sensitivity, respectively. A549 cells were transduced with rSIV.F/HN or were left untransduced as controls. Cells were harvested 4 hours after gene transfer to assess whether RNAScope (using a lentiviral vector-specific sequence) was able to detect vector genomes in transduced cells. Vector genomes were detectable in transduced, but not untransduced cells (figure 1) and we are currently evaluating the technique in lungs of mice and sheep models. Conclusion Both DD-RT-PCR and RNAScope hold promise for assessment of safety and efficacy in the upcoming lentivirus CF gene therapy trial.