(405a) A Platform CRISPR-Cas12a Assay for Rapid Quantification of Adeno-Associated Virus Vector Genome Titers

Gene therapy has experienced a meteoric rise in clinical interest as a next generation therapeutic modality for treatment of genetic diseases. Adeno-associated virus (AAV) vectors are one of the most widely used gene therapy delivery platforms because of their non-pathogenicity, ability to target multiple tissue types, and stable gene expression. AAV clinical demand has recently started to outpace current vector manufacturing capability due to several manufacturing challenges. Further complicating manufacturing productivity issues is a lack of access to innovative process analytical technology (PAT) for efficient process monitoring. One of the most prominent analytical challenges is the fast and accurate determination of the total vector genome, a critical quality attribute required to be monitored for clinical production. Here, we designed and developed a CRISPR-Cas12a based vector genome quantification assay for at-line AAV quantification, delivering accurate determination of the vector genome concentration within 30 minutes. We constructed the CRISPR-Cas12a assay to specifically target the conserved inverted terminal repeats (ITRs) of the AAV genome as a potential platform AAV diagnostic approach. The CRISPR assay accurately quantified the genome titer of 2 different genomic constructs encapsidated in 3 common AAV serotypes (AAV2, AAV5, and AAV6). Under the optimized conditions, the assay detected each AAV below 5e8 vg/mL with >1 log working range within 30 minutes. This new PAT drives down the time-to-result for gene therapy process analytical technology while simplifying the vector genome titration workflow with a highly flexible assay format, which represents a new potential tool for vector analytical characterization.