Insight and Development of Advanced Recombinant Adeno-Associated Virus Analysis Tools Exploiting Single-Particle Quantification by Multidimensional Droplet Digital PCR

Abstract

Recombinant adeno-associated virus (rAAV) has become the most widely used vector in the gene therapy field with hundreds of clinical trials ongoing and already several products on the market. AAV's physicochemical stability, and the various natural and engineered serotypes allow for targeting a broad range of cell types and tissue by diverse routes of administration. Progressing from early clinical studies to eventual market approval, many critical quality attributes have to be defined and reproducibly quantified, such as AAV stability, purity, aggregates, empty/full particles ratio, and rAAV genome titration. Droplet digital PCR (ddPCR) is becoming the tool of choice to perform absolute quantification of rAAV genomes. In the present study, we have identified critical parameters that could impact AAV titration and characterization accuracy, such as Poisson distribution confidence interval, primers/probe position, and potential aggregates. Our work presents how ddPCR can help to better characterize AAV vectors on the single particle level and highlights challenges that we are facing today in terms of AAV titration.

Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR

“…One method uses the linkage concentration derived from the excess number of double positive droplets relative to the number of double positive droplets expected at a given concentration by random encapsulation of both the FAM and HEX target sequences [ 17 ]. An alternative method uses the percentage of double positive droplets calculated from the three positive droplet clusters (FAM+HEX-, FAM-HEX+, and FAM+HEX+) as either a concentration [ 9 ] or as the droplet number [ 20 ] for each positive droplet cluster. We used three control sample types (female DNA, plasmid DNA and synthetic DNA) to compare the two methods ( Fig 9 ).…”

“…We evaluated two different methods for determining the genome integrity (i.e., linkage) using three different sample types with varying expected genome integrity or linkage values ( Fig 9 ). The method based on the percentage of double positive droplets using either the concentration [ 9 ] or droplet number [ 20 ] was highly dependent on the concentration of the template for a concentration range covering approximately 100–5000 copies/μL and generally did not agree with the expected genome integrity of three different sample types with different genome integrity. The double positive droplet percentage method produced the expected genome integrity for a plasmid sample that was 100% linked but had little selectivity for any of the other conditions tested, which included targets that were on separate chromosomes and should have been unlinked with a genome integrity of 0%.…”

“…The female DNA and synthetic gBlock DNA used HaeIII (New England Biolabs, R0108L) and the plasmid DNA used either HindIII (New England Biolabs, R0104L) or MspI (New England Biolabs, R0106L). The genome integrity or linkage between two assays was determined using either the percentage of double positive droplets or the linkage percentage based on the excess number of double positive droplets relative to the number of double positive droplets expected by random co-encapsulation of two targets at a given template concentration [ 9 , 17 , 20 ].…”

Thermostability and in vivo performance of AAV9 in a film matrix

“…For AAV vectors, these have traditionally fallen into two classifications: those that characterize the number of physical particles or genomes present in a preparation and those that assess vector potency [38][39][40][41] . To date, PCR-based assays are the most widely used and accepted method for quantification of AAV vectors as they are simple and robust under ideal conditions [42][43][44] . However, the number of virus genomes present in a given production lot, cell, or tissue sample does not fully demonstrate that the virus is functional.…”

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing

“…It has also been shown that the use of a multi‐dimensional PCR assay to measure rAAV genome titer can provide insight into what percentage of amplicon‐containing capsids may have full versus partial genomes. Two‐plex digital droplet PCR (ddPCR) assays targeting combinations of the promoter, transgene, and polyadenylation (polyA) tail have shown that only 50−70% of single‐stranded DNA containing capsids in rAAV samples contain double positive genomes, while up to 96% of plasmid DNA controls contained both targets, suggesting that multi‐amplicon PCR analysis can discern full from partial capsids (Furuta‐Hanawa et al, 2019; Hayes & Dobnik, 2022; Zanker et al, 2022).…”