Quality control for adeno-associated virus genome preparations using nanopore sequencing

Adeno-associated viruses (AAV) vectors are a promising gene delivery vehicle used in gene therapy. They are replication-deficient viruses containing a 4.8 kb single-stranded DNA genome. AAVs are increasingly being used for in vivo DNA delivery, with five AAV-based gene therapies already approved by the FDA.​ Identifying vector genome quality issues that can arise during the manufacturing process, such as mutations, truncation hotspots, and potential contaminants, is critical.

Here we describe an AAV workflow, developed by Oxford Nanopore Technologies, for Quality Control (QC) and validation of rAAV preparations and the plasmids used to create them. Nanopore sequencing can generate reads that span the entire AAV genome, from ITR to ITR, with the ability to sequence through GC-rich regions, capture vector heterogeneity, and identify nucleic-acid impurities from plasmid and cell line DNA. This allows for the full characterisation of the composition of rAAV vector preparations, assisting with the production of safe and effective vectors for downstream therapeutic uses.