799. Initial Characterization of a Super-Permissive Cell Line for Adeno-Associated Virus (AAV): Microarray-Intimated CK2 Helps AAV Replication

Abstract

Adeno-associated virus (AAV) infection is negatively associated with cervical cancer and is being used as a human gene therapy vector. However, AAV is very selective as to the specific conditions under which it replicates. The identification of cellular genes involved in AAV replication will allow for a better understanding of AAV's molecular biology and AAV's oncoprotective phenotype, as well as allow for the easier generation of recombinant (r)AAV for gene therapy. Here we identify a primary cervical cancer cell line, PT3, which allowed for 10 fold higher AAV DNA replication levels than other isolates and normal primary keratinocytes. Microarray gene expression comparative analysis of PT3 cells to others identified many differences in gene expression. Casein kinase 2 (CK2) was found to be the most highly expressed protein kinase in PT3 cells. Following this lead CK2 stimulated AAV DNA replication in normal keratinocyte-based organotypic epithelial rafts (skin) both as an expressed cDNA ( subunit) or as transfected protein. CK2 also phosphorylated the AAV encoded Rep78, major replication/regulatory protein in vitro. These data suggest that CK2 may be a cellular helper gene for AAV, possibly through phosphorylating Rep78. However, the number of genes identified by microarray comparative analysis suggested that, in addition to UP-regulated genes such as CK2, the genes which are DOWN-regulated might also be significant for AAV replication as four fold more such down-regulated genes were identified.