Abstract
We have discovered a novel anti-sense promoter element in nucleotides proximal to the right ITR of AAV serotype 2. Presence of the element was initially detected using vector derived from psub201, a widely-used plasmid for AAV vectorization which carries an infectious wtAAV2 clone engineered such that the left ITR is replaced with the right ITR and 46 nucleotides (nt) of proximal AAV2 sequence. While preliminary studies using the psub201-derived vector showed promoter activity originated from a sequence containing 46-nt of wtAAV2 and 59-nt of poly-linker sequence, further investigation demonstrated 105 nucleotides proximal to the right wtAAV2 ITR to be optimal for activity, which was further enhanced by presence of ITR sequence. Demonstration of similar promoter activity in a phylogenetically-related AAV serotype (AAV7) but not a more distantly related serotype (AAV5) indicates potential conservation of the activity amongst related AAV, which may be important for the virus cycle. Activity of the element is restricted to cells of hepatic origin but also operates in a lung sarcoma cell line (A549). Our findings take on new significance in light of a recent study by Nault and colleagues (Nault et al. 2015 Nature Genetics) who have associated genomic integration of AAV with HCC in 11/193 patients. Cloncal expansion of AAV integration events occurred in known drivers of carcinogenesis leading to gene dysregulation at the site of integration. A striking feature of the study is that only a fragment of the AAV genome was integrated in the tumor genome with one patient showing a 1442 bp fragment spanning the rep-cap junction while the remaining 10/11 tumors contained fragments (201-1975 bp) from the 3’ genome of which all contained the 105-nt element described in the current report. Conclusions drawn from the Nault study that AAV is involved in insertional mutagenesis have caused some contention in the field prompting two editorials with alternative interpretation of the data (Buning and Manfred 2015 Mol. Ther.; Berns et al. 2015 Human Gene Therapy). While the Nault report requires further scrutiny and confirmation, the nature of the findings may have implications for AAV gene therapy, especially in the liver over the longer term. Our findings delineating cell-restricted transcriptional activity from the 3’ promoter element in AAV is of general interest to the fields of AAV biology and vectorology but, more importantly, are of specific significance to the HCC controversy and may provide insight and resolution over the matter of AAV-associated HCC.
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