Zhongya Wang


December 2007

Document Type


Degree Name



Dept. of Cell and Developmental Biology


Oregon Health & Science University


Recombinant adena-associated virus (AA V) vectors are mostly episomal and rarely integrate into the host genome. This feature of random integration poses significant safety concerns regarding the use of AA V in clinical gene therapy. When integration does occur, AA V can integrate randomly throughout the genome. For the purpose of gene therapy for genetic diseases, an integrating vector capable of site-specific integration would be ideal. In our lab, we have constructed an AA V vector in which a human fumarylacetoacetate hydrolase (Fah) expression cassette is flanked by ~ 2 kb of homologous rDNA in the region of the I-Ppoi site. We hypothesized that the AA V vector could be targeted to this location by homologous recombination facilitated by the presence of a double strand DNA break created by I-ppol. To prove our hypothesis, Fah1 - mice were injected with high dose of viral particles (3xl0 11 vg) and phenotypic correction was measured by weight gain after NTBC withdrawal. Adult Fah_ 1 _ mice injected with a dose of3xl0 11 particles (high dose) gained weight after NTBC withdrawal while control mice died after 4-6 weeks. lxl09 AA V8-rDNA-Fah (low dose) rescued Fah_ 1_ mice with initial weight loss, followed by weight gain. Next, the hepatocytes of weightstabilized mice were serially transplanted into secondary Fah_ 1_ recipients. All secondary recipients displayed weight gain after transplantation indicating the presence of a stably integrated Fah expression cassette. Site-specific junction PCR was done to confirm the presence of our hFah expression cassette within the rDNA. In addition, junction PCR would enable us to differentiate homologous recombination events from non-homologous end joining and thus was applied to all the mice including primary mice, transplanted mice, and neonatal mice injected with our AA V -rD N A- Fah. Site-specific junction Wang, Z, AA V based site-specific integration in vivo fragments were detected in all the mice, including neonatal mice. The sequence results of the junction fragments generated by site-specific PCR exactly matched the predicted sequence. Finally, a dose dependent comparison between AA V2-rDNA-Fah and AA V2-Fah showed that AA V2-rDNA-Fah can rescue the Fah1- mice at significantly a lower dose (about 1/30-1/10) compared to the dose required for AA V2-Fah. AA V8-rDNA-Fah is able to rescue Fah_ 1_ mice at a dose of 1x109 . Our strategy was also tested in a hemophilia B mouse model. AAV-rDNA-hFIX was constructed and injected into wild type mice. One month after injection, mice underwent partial hepatectomy (PHx) to remove most of the episomal AA V genome. Surprisingly, the post-PHx and pre-PHx ratio of hFIX level was significantly higher using AAV8-rDNA-hFIX and AAV2-rDNA-hFIX (40-50% and 70-80% respectively), compared to only 5-10% after PHx with conventional AA VhFIX. The results are promising and may be of potential use in human gene therapy trials for hemophilia given the stable integration achieved with high efficiency. Surprisingly, we also detected evidence of site-specific integration systemically in other tissues such as the lung, heart, muscle and kidney in addition to liver as early as 3 days post injection. This highly suggests that AAV8-rDNA is capable of integrating systemically in vivo. This is the first report of site-specific integration in an organ system other than liver. This finding is significant for gene therapy of certain diseases that systemically affect patients. It is possible that with an appropriate tissue-specific promoter or with different AA V serotypes, AA V -rDN A can be specifically used in other tissues. Thereby, our AA V -rDNA vector is superior to currently existing AA V vectors due to its ability to integrate site-specifically within ribosomal DNA with high efficiency at a low rescue dose. Our novel vector opens the possibility for a new strategy for gene delivery for use in clinical gene therapy trials.




School of Medicine



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