Tracking particle-encapsulated DNA across the anion-exchange
chromatography fractions of recombinant adeno-associated virus using
droplet digital PCR and high-throughput sequencing
Abstract
[Introduction] Safe and effective adeno-associated virus vectors are
essential for gene therapy. Particle heterogeneity, specifically
particle DNA of varying types and sizes, significantly affects
recombinant adeno-associated virus (rAAV) performance. Previous studies
have identified particle-associated DNAs; however, the specific DNA
composition of these mixed populations remains poorly understood. This
study aimed to investigate the DNA composition of the isolated
subpopulations of rAAV particles obtained through anion exchange (AEX)
chromatography. [Methods] rAAV2-ZsGreen1 particles were fractionated
on an AEX column, resulting in 12 distinct fractions. We analyzed the
DNA composition of these fractions using droplet digital PCR and MiSeq
to identify the incorporated DNA heterogeneity in them. [Results]
Our findings illustrated a clear trend in which DNA content increasing
in fractions was associated with an increased rAAV genomic DNA ratio of
total particle DNA. The particle DNA content increased significantly
across fractions from Peak 1 to Peak 2, showing approximately 30,000-
and 5,000-fold increases for ZsGreen1 (rAAV genome) and ampR (plasmid
impurity), respectively. Notably, in the empty particle subpopulations,
the rate of detectable DNA molecules was lower than one DNA fragment per
100 particles, with Inverted Terminal Repeat (ITR) sequences being the
most prevalent. [Conclusions] With the elucidated profile of
particle DNAs, this study provides detailed information on particle
heterogeneity, shedding light on empty and partial particle formation
and impurity DNA incorporation.