• Preparation and Construction of rAAV(ss) GNR & dPCR Detection System: The recombinant single-stranded rAAV genome copy number reference material (rAAV(ss) GNR) of serotype AAV843 was successfully prepared by a three-plasmid transfection system combined with ultracentrifugation-based purification. A highly sensitive and reproducible genomic titer detection system based on digital polymerase chain reaction (dPCR) was established. • Comprehensive Quality Validation and Functional Characterization: The reference material showed intact genomic structure with consistent titers of 8.7×10 8 vg/mL in both inverted terminal repeat (ITR) and gene of interest (GOI) regions. It met purity standards verified by SEC-HPLC, CE-SDS, etc., and functional requirements such as TCID 50 and cell viability. • Multi-center Collaborative Validation and Stability Confirmation: The GNR and standardized detection protocols were distributed to 11 gene therapy enterprises and 1 testing institution. Multi-platform evaluations showed that the 95% confidence intervals of titers were 8.6×10 8 –8.8×10 8 vg/mL for ITR and 8.6×10 8 –8.9×10 8 vg/mL for GOI. The material remained stable after storage at ≤−60°C for 18 months. • Application Value for Standardization in Gene Therapy Industry: The rAAV GNR and its supporting detection system provide core tools for quality control and dosage standardization of gene therapy products, demonstrating application potential in areas such as rAAV genomic titer method development, sample stability monitoring, and test result correction, and promoting the standardization of industry detection criteria.
Bi et al. (Fri,) studied this question.