Using digital PCR to characterise viral vector genomes

Viral vectors are an effective gene transfer method to modify a wide range of cells and tissues to produce thera-peutic products to treat or prevent diseases. Before they can be introduced to the host, their safety and effective-ness must be determined by analysing critical quality attributes such as their identity, purity, potency, safety, and stability. Current analytical techniques are limited with low-throughput, large sample requirements, and poorly un-derstood measurement variability. This presentation will focus on the development of a digital PCR (dPCR) meth-od to characterise the genome of AAV particles (modified adeno-associated viruses). The focus is on identification and quantification of complete genomes (correct), partial genomes (incorrect) or empty (non-function) genomes within the capsid of the viral particle. Briefly, dPCR sub-divides a quantitative PCR reaction by limiting dilution into thousands of sub-nanolitre reactions, termed partitions. PCR cycling is performed, each partition “read” and assigned as positive or negative based on the presence or absence of fluorescence, respectively. The copy number concentration is estimated using the pro-portion of positive partitions, with the application of Poisson statistics to account for partitions that initially con-tained more than one target. This approach means that a standard curve is not required to estimate the copy num-ber concentration. Furthermore, multiplexing enables regions along the full length of the vector genome to be tar-geted and so enable quantification of full length genomes as those that are contained in a partition that is positive for all targets.