500. Biology of Novel AAV Serotype Vectors in the Liver Directed Gene Transfer to Nonhuman Primates
Authors: Guangping Gao, You Lu, Roberto Calcedo, Rebecca Grant, Julio Sanmiguel, Lili Wang, Julie Johnston, Joanita Figueredo, Martin Lock, James M. Wilson
Journal: Molecular Therapy (2005)

Top of pageAbstract
Recent advance in the AAV vectorology has led to the isolation of novel primate AAV vectors that can achieve efficient and stable gene grafting in the murine liver. Critical to further development of AAV vectors for potential clinical applications is their evaluation in large animal models including nonhuman primates (NHPs). In the present studies, we evaluated sero-epidemiology of novel AAV serotypes and their vector biology in NHPs. We discovered that while preexisting immunity to novel NHP AAV derived vectors was highly prevalent in NHP populations, frequency and potency of neutralizing antibodies were higher in rhesus than cynomolgus macaques. Sero-positives to human AAVs were low for AAV2 as expected but unexpectedly high for AAV5. We packaged an AAV2 based vector genome with the capsids from AAVs 2, 5, 7 and 8 for the liver directed either the rhCG or rhEpo gene transfer to rhesus or cynomolgus macaques.

In the first study where rhCG was used as a reporter, we observed sustained one-log higher rhCG levels in the AAV7 vector treated rhesus macaques as compared to those in the AAV2 and AAV5 cohorts. However, rhCG expression profiles in the AAV8 animals were more complicated due to the presence of preexisting B cell immunity that either directed to or cross-reacted with AAV8. The study was repeated in cynomolgus macaques where serological prevalence of NHP AAVs is lower for liver directed rhEpo gene transfer. Our data revealed that the new serotype vectors could target liver 10 to 100 times more efficiently than AAV2 vector as determined by Southern blot and TaqMan PCR, which resulted in 10 to100-fold higher circulating rhEpo in the early phase of the gene transfer. However, rhEpo levels in the other serotype groups rapidly declined to and stabilized at the levels that were similar to those in one AAV2 animal and 10-fold higher than those in the lower expressing AAV2 animal. Overall, AAV vector mediated gene transfers to the NHP liver caused little toxicity as measured by liver function tests, cytokine profiling and histopathological analysis.

In another study, we attempted to further enhance biopotency of novel rAAVs in NHPs by using self-complementary AAV (dsAAV) developed by Samulski and Xiao's groups. Our preliminary data from the early time points revealed that dsAAV7CBrhCG displayed 20 to 200-fold higher gene expression than the same vector in the ss form with a rapid on set, approached the peak by day 3. Remarkably, rhCG levels per genome in the dsAAV7CBrhCG treated animals were higher than those received ssAAV7TBGrhCG. Long term comparison of ds and ssAAV7 vectors in NHPs remains to be examined.

Our studies suggest that novel NHP AAV mediated gene transfer to the NHP liver is efficacious. But efficiency and stability of novel could be influenced by the preexisting immunity to the vector capsids. In addition, sustained high-level expression of circulating transgene product such as rhEpo that is not naturally produced by liver might be limited by the liver's capacity to process and secret such a protein.