Zaidi, H. | Search bioRxiv

bioRxiv2025

AAVone: A Cost-Effective, Single-Plasmid Solution for Efficient AAV Production with Reduced DNA Impurities

Currently, the most common approach for manufacturing GMP-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. The requirement for all three plasmids to be transfected into a single cell and the necessity for high quantities of input plasmid DNA, limits AAV production efficiency, introduces variability between production batches, and increases time and labor costs. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone. In this system, the adenovirus helper genes (E2A, E4orf6, and VA RNA), packaging genes (rep and cap), and the vector transgene cassette are consolidated into a single compact plasmid with a 13-kb backbone. The AAVone system achieves a two- to four-fold increase in yields compared to the traditional triple-plasmid system. Furthermore, the AAVone system exhibits low batch-to-batch variation and eliminates the need for fine-tuning the ratios of the three plasmids, simplifying the production process. In terms of vector quality, AAVs generated by the AAVone system show similar in vitro and in vivo transduction efficiency, but a substantial reduction in sequences attributed to plasmid backbones and a marked reduction in non-functional snap-back genomes. In Summary, the AAVone platform is a straightforward, cost-effective, and highly consistent AAV production system - making it particularly suitable for GMP-grade AAV vectors.

bioengineering↗

bioRxiv2017

Second-generation method for analysis of chromatin binding using formaldehyde crosslinking kinetics

Formaldehyde crosslinking underpins many of the most commonly used experimental approaches in the chromatin field, especially in capturing site-specific protein-DNA interactions. Extending such assays to assess the stability and binding kinetics of protein-DNA interactions is more challenging, requiring absolute measurements with a relatively high degree of physical precision. We previously described an experimental framework called CLK, which uses time-dependent formaldehyde crosslinking data to extract chromatin binding kinetic parameters. Many aspects of formaldehyde behavior in cells are unknown or undocumented, however, and could potentially impact analyses of CLK data. Here we report biochemical results that better define the properties of formaldehyde crosslinking in budding yeast cells. These results have the potential to inform interpretations of standard chromatin assays including chromatin immunoprecipitation, and the chemical complexity we uncovered resulted in the development of an improved method for measuring binding kinetics using the CLK approach. Optimum conditions included an increased formaldehyde concentration and more robust glycine quench conditions. Notably, we find that formaldehyde crosslinking rates can vary dramatically for different protein-DNA interactions in vivo. Some interactions were crosslinked much faster than the time scale for macromolecular interaction, making them suitable for kinetic analysis. For other interactions, we find the crosslinking reaction occurred on the same time scale or slower than binding dynamics; for these it was in some cases possible to compute the in vivo equilibrium-binding constant but not on- and off-rates for binding. Selected TATA-binding protein-promoter interactions displayed dynamic behavior on the minute to several minutes time scale.

biochemistry↗