95. Incorporation of AAV Serotype 2 Viral Protein 2 into Other AAV Serotype Capsids

Abstract

It was previously documented that large ligands inserted after residue 138 in the adeno-associated virus serotype 2 (AAV2) viral protein 2 (VP2) is tolerant to produce nearly wild-type levels of recombinant AAV2 (rAAV2) vectors (J Virol. 78(12):6595-609, 2004). Furthermore, insertion of enhanced green fluorescent protein (EGFP) into VP2 resulted in a rAAV2 particle, whose trafficking could be temporally monitored using confocal microscopy. Here, we examined whether AAV2 VP2 (2VP2) could be incorporated into other AAV serotype capsids during viral production.A pcDNA3.1-based plasmid was generated to over-express only the 5’-EGFP-tagged 2VP2 (EGFP-2VP2), but not other viral proteins, such as AAV2 Reps, VP1, or VP3. Then, rAAV1~10 vectors were produced in HEK293 cells using PEI-mediated, quadruple-plasmid transfection. The four plasmids for each rAAV vector included a packaging plasmid that contains AAV2 rep genes and a corresponding cap gene, a helper plasmid that provides essential genes from adenovirus, an ITR-containing plasmid that carries a reporter gene encoding firefly luciferase (Fluc), and the pcDNA3.1-EGFP-2VP2 plasmid. The expression of corresponding VP2 in each serotype capsid was not blocked. Purified viral stocks were separated by SDS-12% acrylamide gel electrophoresis and analyzed by Western blot assays with the B1 antibody or EGFP antibody. The results indicated that the EGFP-2VP2 protein was successfully inserted into all tested rAAV vectors, except for rAAV5. Approximately one-tenth to one-half of the total VP2 was substituted by EGFP-2VP2, depending on the serotype. Next, we characterized the novel rAAV2 and rAAV3 particles that had EGFP-2VP2 insertions. Four vectors (WT-rAAV2, rAAV2-EGFP-2VP2, WT-rAAV3, rAAV3-EGFP-2VP2) carrying the fluc gene were used to transduce a human hepatocellular carcinoma cell line, Huh7, under identical conditions in vitro. There was no difference in the transgene expression between the WT and EGFP-2VP2-inserted vectors. Furthermore, when heparin was added in the cell culture, the transduction efficiency of both rAAV2 vectors was inhibited in a dose-dependent manner. On the other hand, the transduction efficiency of rAAV3-2VP2-EGFP vectors showed a similar pattern to that was previously reported for WT-rAAV3 vectors, in which low doses of heparin significantly enhanced rAAV3 transduction, but high doses inhibited it. The tropism of rAAV2-EGFP-2VP2 vectors was further characterized in vivo following tail-vein injection of Balb/c mice and showed no difference compared with the WT-rAAV2 vectors. Finally, the trafficking of both rAAV2-EGFP-2VP2 and rAAV3-EGFp-2VP2 was temporally monitored using confocal microscopy.In summary, we have demonstrated an easy method that can be used to insert large peptides into most AAV serotype particles. This system may provide significant information to study rAAV transduction.