290. A High-Throughput, Sensitive, Accurate and Reproducible Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate
Authors: Jianzhong Ai, Raed Al-Abbasee, Guangping Gao
Journal: Molecular Therapy (2016)

Recombinant adeno-associated viruses (rAAVs) have become an attractive tool for the delivery of therapeutic gene products. Increasing interest and application of rAAV vectors in basic and clinical research has urged efforts to improve AAV production quality and yield. Currently, standard procedures call for genome titration of purified vectors to assess yield at the endpoint of vector production. Mid-production (in-process) methods to gauge AAV yield is severely lacking. In addition, the need to multiplex the production of small-scale AAV batches (e.g. novel serotypes/variants screening) may become cost-prohibitive if vector purification steps were required for hundreds of candidate viral genomes. The high cost of downstream processing and purification during large-scale AAV production can be significantly reduced if low-yield vector batches were identified with earlier monitoring steps during vector production. To meet this need, we have established an enhanced qPCR method for rAAV genome titration in crude lysate. In brief, critical parameters call for the elimination of inhibitory factors inherent to cellular lysates that may reduce qPCR efficiency. These factors may include contaminating nucleic acids originating from packaging vector and helper plasmids and cellular genomes and proteins that are residual from the host cell. Lysate samples are thus directly treated with DNase I and proteinase K in 96-well plates to remove cellular/plasmid DNAs and abundant proteins, followed by TaqMan quantitative PCR analysis on Dnase I-resistant vector genomes. To determine treatment efficiencies, we also quantified the copy numbers of vector plasmids diluted in non-transfected crude lysate before and after treatment. We demonstrate that plasmid templates are completely removed by DNase I treatment and qPCR efficiency is markedly enhanced by proteinase K. This enhanced qPCR method is proven to be highly sensitive and reliable, as it can detect as low as 10 copies of plasmid in crude lysate diluent. Furthermore, we demonstrate that this method is scalable for simultaneous vector genome titration by testing the yields of 13 different serotypes of ssAAV and scAAV. Thus, this procedure can be applied to screen large panels of novel serotypes/variants for vector production. Importantly, we show across independent experiments that titer quantification has high inter-experimental reproducibility among multiple batches of samples. In summary, our described method fulfills unmet needs for quantifying vector genomes in crude lysates from both large- and small-scale AAV preparations in a high-throughput, sensitive, accurate, and reproducible manner. This will significantly improve in-process quality control, batch/lot monitoring in large-scale preparations, and good manufacturing practices (GMP) for AAV production - all key for vector manufacturing, as AAV continues to garner use and impact in both basic research and clinical applications.