We seek to continue our analysis of human papillomavirus (HPV) helper gene functions for adeno-associated virus (AAV) replication. AAV is the safest vector for use in human gene therapy and gives the longest reported transgene expression. HPV helper genes can be used to strongly boost rAAV production. In addition, AAV infection limits HPV-associated cervical cancer, one of the most prevalent cancers in the world. Thus, understanding the HPV/AAV relationship is important for multiple reasons. Previously we have identified HPV E1, E2, and E6 as helper genes for AAV replication. We initially concentrated on studying E1 helper function and found that E1 directly binds the AAV Rep78 replication protein and causes increased Rep78-inverted terminal repeat (ITR) DNA binding, increased nickase, and increased covalent attachment biochemistries. These activities are the central roles of Rep78 during AAV DNA replication. Moreover, our preliminary data show that each of the three HPV helper genes strongly improve rAAV production in HEK293 cells in collaboration with the standard adenovirus (Ad) helper gene set. This is important as the production of rAAV is a major problem for its use in human gene therapy. In this application we continue our studies of HPV E1, E2 and E6 helper activities. For E6 we hypothesize that E6 lowers p53 levels, allowing higher AAV replication, as Ad E1B55K and E4orf6 are inadequate in effective reduction of p53 in HEK293 cells. For E1 we hypothesize that an E1 domain in the carboxy-half of the protein, when used alone, has enhanced helper function over that of wild type E1. For E2 we hypothesize two mechanisms of action. First, we have identified an E2-responsive enhancer buried within AAVs capsid gene sequence. We will characterize this enhancer and, in addition, we will optimize E2 motif sequences to result in even stronger E2-responsiveness, preferentially up-regulating the adjacent cap gene. Second, E2 , like E1, directly binds Rep78. We hypothesize that E2, like E1, will also modulate/stimulate important Rep78-ITR biochemical activities, critical for AAV DNA replication. To study HPV's helper functions for AAV we propose the following four aims: Aim #1: we will study the effect of HPV16 E6 on p53 levels in HEK293 cells during rAAV production along with the standard Ad helper gene set. We expect that the E6-related boost in rAAV production will be linked to lower p53 levels. Aim #2: we will characterize, deletion map, and study the use of the E1 sub-protein helper domain in HEK293 cells during rAAV production along with the standard Ad helper gene set. Aim #3: We will characterize the identified E2- responsive enhancer in AAV2 and determine its role in the AAV life cycle. We will map the specific elements of the E2-responsive enhancer by deletion, determine the importance of the E2-responsive enhancer within the context of the full length AAV genome, and generate an enhanced E2-responsive enhancer near the 3'end of the AAV2 cap gene using fully consensus E2 motifs and observe if helper activity, in particular cap mRNA and protein levels, are increased. Aim #4: We will identify the effects of HPV16 E2 on AAV2 Rep78-ITR biochemistry, using EMSA, ATPase, helicase, covalent attachment and nickase assays.

R56AI093695

2011-09-30

2013-09-29