McMaster University

Purpose

This project aims to develop an analytical method to measure transduction efficiency at the single-cell level in a gene-modified, viral-vector based immunotherapy model. The method will employ digital droplet PCR (ddPCR) implemented in polymer microfluidic devices to encapsulate engineered cells with appropriate reagents (lysis and PCR master mix), including vector specific primers and hydrolysis probes, as well primers and probes for the reference gene. The encapsulated droplets will then be subjected to thermal cycling and subsequently arrayed in a monolayer format for fluorescence imaging. Image analysis and count of positive droplets for each fluorophore will allow accurate assessment of the transduction efficiency. This one-step lysis and digital PCR system aims at facilitating and streamlining the assessment of the viral transduction efficiency, an important step in the manufacturing of engineered T cell therapies.