Guise, M. | Search bioRxiv

bioRxiv2025

Fluctuations in AAV genome quantification via digital PCR affected by heat-based virion lysis

Adeno-associated virus (AAV) vectors are central to gene therapy, making precise genome quantification essential for product quality and dose determination. We systematically assessed digital PCR (dPCR) on the QIAcuity platform to quantify recombinant AAV2 and AAV8 genomes, examining how assay design, amplicon length, and heat-based sample preparation affect results. Across multiple genomic targets, shorter amplicons consistently yielded higher copy numbers than longer ones leading to a quantification deviation of up to [~]48%. The results point to heat-induced genomic fragmentation as the main cause of the observed effect. These findings highlight that dPCR-based AAV quantification is highly sensitive to amplicon length and sample preparation. We propose the use of the employed multitarget assay set to evaluate AAV genome extraction procedures, the quality of AAV genomic DNA extracts, and ultimately the AAV genome integrity.

molecular biology↗

bioRxiv2025

Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production

Cost-effective and scalable production is critical for advancing the clinical translation of adeno-associated virus (AAV)-mediated gene therapy. The widely used transient transfection method using plasmid DNA (pDNA)-loaded transfection particles for AAV production faces technical challenges due to instability of the particles and the concentration limits for particle preparation, hindering reproducibility and scalability. Here, we report a streamlined and scalable strategy to generate shelf-stable, highly concentrated pDNA/poly(ethylenimine) (PEI) transfection particles. By incorporating trivalent citrate ions in the dilution buffers, we kinetically modulate electrostatic complexation to achieve uniform nanoparticle assembly and prevent aggregation at high concentrations. This enables a tenfold increase in pDNA concentration in stabilized transfection particles from a typical range of 10-20 g/mL to 200 g/mL, while reducing the required dosing volume from 5-10% to 0.5% of the cell culture medium. The particle assembly process is robust to changes in mixing scale and timing and is compatible with standard workflows. We demonstrate equivalent AAV production efficiencies to standard methods and consistent performance in various production scales, which confirms the practical utility of this assembly method in developing robust, scalable, and cost-effective AAV manufacturing processes.

biochemistry↗