102. Method Validation for Quantification of Recombinant Adeno-Associated Virus 5 in Mouse Genomic DNA Using Real-Time PCR

The recombinant adeno-associated viral (rAAV) vectors are one of the most promising tools in human gene therapy because of their excellent safety profiles. Recently, one of these, rAAV serotype 5 (rAAV5) has attracted attention as a therapeutic vector by many researchers. To develop rAAV5 vector for treatment in clinical trials, it is necessary to perform GLP compliant preclinical toxicity and biodistribution studies in animals. Quantitative real-time PCR (qPCR) technique is gold standard for quantification of gene therapy products and also has been used as the most sensitive method for bioanalysis of other rAAV vectors. In present study, an analytical method by qPCR was validated for determination of rAAV5 encoding human transgene in mouse genomic DNA. The validation was conducted to evaluate specificity, linearity, accuracy, and precision. There were no interfering reacts with the targeted sequence between true positive and negative control (no template control) samples. The correlation coefficient of rAAV5 in mouse genomic DNA was 0.9995 (1/x2 weighted) over a concentration range of 100 to 10,000,000 copies/ug. This method was accurate and precise verified by intra- and inter-batch analysis. The LLOQ for rAAV5 in mouse genomic DNA was 100 copies/ug, measured with acceptable accuracy and precision. Thus, the analytical method was quantifiable, linear, accurate, and precise. In conclusion, the validation method for rAAV5 in mouse genomic DNA has been successfully performed and will be also useful for further preclinical toxicity and biodistribution studies.