S82 Quantification of mRNA and protein from single cells for cystic fibrosis gene therapy

Introduction and Objectives Gene therapy for cystic fibrosis is approaching clinical trials. Using a lentiviral vector pseudotyped with Sendai virus fusion and hemagglutinin-neuraminidase proteins, the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be inserted into airway cells leading to the long-term production of functional CFTR protein in animal models. To evaluate the success of a therapy in the clinic, methods are needed that can accurately and sensitively quantify the number of cells transduced, measure RNA, and measure protein from limited human clinical trial samples. Here we develop RNAscope, reverse transcriptase droplet digital PCR (RT-ddPCR), and digital proximity ligation assay (dPLA) techniques that can be used to detect and quantify lentivirus RNA and Enhanced Green Fluorescent Protein (EGFP) from single cells of tissues transduced in vitro, ex vivo, and in vivo. Methods HEK293T, A549, human air liquid interface cultures, mouse nasal brushings, mouse airway, and human nasal epithelial cells were transduced with lentivirus encoding EGFP and the woodchuck hepatitis post-transcriptional regulatory element (WPRE). Following transduction, cells were spun and fixed onto slides for RNAscope using a probe against WPRE. Alternatively, transduced cells were single-cell flow-sorted into 96-well plates. RNA and protein were measured from the same cell. For RNA quantification, one-step RT-ddPCR was performed with a probe against WPRE. For protein quantification, dPLA used oligonucleotide conjugated antibodies against GFP. Results RNAscope successfully stained for WPRE in A549, mouse airway cells, and human nasal brushings. In mouse airways cells, RNAscope measured a median of 33.9% and 24.9% of WPRE+ cells 7 and 28 days after transduction, respectively. RT-ddPCR and dPLA successfully detected WPRE containing RNA and EGFP from all tissues analysed. In mouse nasal brushings, RT-ddPCR measured a median of 951.0 WPRE RNA copies/cell and dPLA measured a median of 9908 dPLA templates/cell 28 days after transduction. Additionally, EGFP protein levels significantly correlated to EGFP fluorescence levels measured during cell sorting (R2 = 0.94, p Conclusions RNAScope, RT-ddPCR, and dPLA can successfully detect lentiviral RNA and EGFP from multiple tissue sources. These methods could prove useful for testing therapeutic vector transduction efficiency both in pre-clinical studies and clinical trials.