Dept. of Cell and Developmental Biology
Oregon Health & Science University
AAV is one of the most promising gene therapy vectors. There are several clinical trials with AAV vectors for different genetic disease. For gene therapy in genetic diseases, the diseasesâ nature requires persistent expression of the transgene. One of the main disadvantages for AAV vectors is that a vast majority of AAV vectors in vivo exist as episomal, as the integration frequency is very low. In this dissertation, we engineered a new integrating AAV-rDNA vector. We firstly used the HT1 disease model to characterize this vector. At the DNA level, AAV-rDNA-FAH were found to give 10-30 times more integrated viral genome than sizable control AAV-stuffer-FAH at two different doses by real time PCRs. At protein level, we used anti-FAH immunostaining to stain the AAV infected liver and found that AAV-rDNA-FAH gave 18 times more FAH positive nodules than control vectors. At functional level, AAV-rDNA-FAH vectors were able to rescue HT1 mice, which will die without any treatment, at 10-30 times lower dose than comparable control. Importantly, rescued Fah[superscript -/-] mice all displayed normal liver function test as similar to wild type mice no matter rescued by high dose or low dose. These data provide evidence that AAV-rDNA vectors gave about 10-30 times more viral vector integration with functional transgene expression. These results have been confirmed in two different disease models, HT1 mouse model and hemophilia B mouse model. In this dissertation, we also characterized that the minimum homologous rDNA length required for efficient integration will be 750 bp. In both disease models, we detected the existence of site-specific integration. By LAM-PCR, shuttle vector strategy, PCR base next generation sequencing, we identified different sets of integration sites of AAV-rDNA vectors, all of which show significant enrichment of AAV-rDNA integration in rDNA repeats by more than 10 times. This provided consistent results with DNA level, protein level, and functional level data generated in two disease models. Taken together, we have designed an efficient integrating vector, which gives 10 times more integration with functional transgene expression. When treated for genetic liver diseases or other genetic diseases, which required persistent transgene expression, this vector should be considered and could provide a significantly better therapy than regular AAV vectors.
School of Medicine
Wang, Zhongya, "A newly designed AAV-rDNA vector for persistent therapy in genetic liver diseases" (2012). Scholar Archive. 750.