Several recombinant adeno-associated virus-based gene therapy products have been developed recently. The vector genome (VG) titer is a critical quality attribute associated with the clinical dosing of these products and, thus, requires accurate quality control measures. Typically, VG titers are measured by quantitative polymerase chain reaction (qPCR). Droplet digital PCR is more reliable than qPCR and a powerful analytical tool for quantifying genome copies with high accuracy and precision; however, VG titers cannot be correctly quantified without the appropriate preparation of analytical test samples. In this study, we systematically assessed the role of each component and treatment comprising DNase treatment for free and residual DNA, DNase inactivation, and single-stranded DNA extraction from capsids in VG titration results during pre-analytical sample preparations. Incubation near the single-stranded DNA-release temperature decreased the number of recombinant adeno-associated virus genome copies. Moreover, we developed a simplified three-step pre-analytical procedure with concurrent DNase inactivation and single-stranded DNA extraction at a much higher temperature than the release temperature. Developing an analytical procedure for recombinant adeno-associated virus genome titration by droplet digital PCR based on release temperature is a science- and risk-based approach that would improve quality control testing of recombinant adeno-associated virus-based gene therapy products.

Advances in gene therapy have led to a wide range of gene therapy products. Hence, it is challenging to use standard non-clinical safety tests, which are typically used to evaluate chemically synthesized products. Therefore, a case-by-case safety evaluation based on the unique characteristics of each product is necessary. In Japan, the fundamental considerations for evaluating the non-clinical safety of gene therapy products are outlined in “Ensuring the Quality and Safety of Gene Therapy Products”. Notably, when designing non-clinical safety studies, it is essential to consider factors such as the efficiency of vector transduction, tissue/cell tropism of the vector, and species differences in the expression level and biological activity of transgene products in the target tissue/organs. However, it is also crucial to understand the limitations of animal studies that arise due to species differences and to assess the safety of expression vectors and expressed genes based on a thorough literature review of their biological properties. The aims of this review are to present non-clinical safety evaluation approaches for gene therapy products as outlined in the Japanese guidelines and key considerations for appropriate non-clinical safety evaluation of in vivo gene therapy products.

Experimental animal models play an essential role in the study of pathogenic mechanisms and development of novel therapies for diseases. Many disease models are developed by introducing genetic factors and environmental factors of disease onset through drug administration, surgery, or genetic modification that results in disease phenotypes. In addition, higher animals such as non-human primates are useful for investigating diseases whose phenotypes cannot be produced in mice, etc. In monkeys, some strains have diseases similar to humans by spontaneous onset, as a result of repeated generational changes over time. Disease models must have high reproducibility, extrapolation to humans, and applicability. In this review, we summarize recent advances in the development of mouse models of Alzheimer’s disease and the studies that utilize them.