Keith Connolly from Modalis Therapeutics spoke at the Biopharma Day in Boston. The title of the session was “Nanopore sequencing and functional screening of AAV genomes for optimal production and function.” Modalis is developing a platform for gene expression modulation. They have been packaging promoters, transgenes, poly(A) signals into AAV genomes. They have been using nanopore sequencing technologies to sequence the AAV genomes they produce. Connolly noted that AAV genomes are small and often contain cargo flanked by ITR sequences with secondary structures. The genomes are single-stranded DNA. A DNaseI treatment is used to extract AAV genomes for sequencing and remove other DNA. It can be challenging to ligate adapters onto the ends of AAVs. The rapid transposome complex prep from ONT was a solution! However, one disadvantage is that reads may not extend the full length of the genome. The ITRs have two different conformations, flip and flop, that were identifiable in the sequencing signals. There were some chimeric alignments from promoter reads. Structure analysis indicated that these sequences had stable structures. Connolly explained that they think there is a mixed population of AAVs with some truncated non-functional genomes. This information was used to screen AAV genomes and identify problematic elements. It is fascinating how even small genomes can have complexities unlocked through sequencing and bioinformatics!
