101. Generation of Improved Recombinant AAV3 Serotype Vectors Using AAV3 ITRS and AAV3 REP Proteins

Abstract

Although recombinant AAV3 serotype vectors were largely ignored previously, owing to their poor transduction efficiency in all cells and tissues examined, our initial observation of their selective tropism for human liver cancer cell lines and primary human hepatocytes (Mol Genet Metabol., 98: 289-299, 2009; Hum Gene Ther., 21: 1741-1747, 2010; Gene Ther., 19: 375-84, 2012), has led to renewed interest in this serotype, since AAV3 vectors and their variants have now proven to be extremely efficient in targeting human and non-human primate hepatocytes in vitro as well as in vivo (Mol Ther., 22: S2, 2014; Mol Ther., 22: S91, 2014; Nature, 506: 382-386, 2014; Hum Gene Ther., 25: 1023-1034, 2014). Previously, it wasreported that the combination of ITR8 with AAV8 capsids (AAV8/8) resulted in vectors that led to at least 2-fold increase in transgene expression in mouse liver, compared with AAV8 capsids pseudotyped with ITR2 (AAV2/8) vectors (Mol. Ther. 11: S156, 2005), and that the ITRs from AAV serotypes 1-6 were interchangeable when they are packaged into AAV8 capsids (AAV1/8 – AAV6/8), but played no role in transgene expression in murine hepatocytes in vivo (J. Virol., 80: 426-439, 2006). In our present studies, we wished to evaluate the relative contribution of the cis-acting ITR from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the recombinant AAV3 vector production and transduction. To this end, we utilized two helper plasmid: pAAVr2c3, which carries rep2 and cap3 genes and pAAVr3c3, which carries rep3 and cap3 genes. Plasmid transfection assays revealed that both AAV2 and AAV3 Rep proteins were expressed at similar levels in the presence of adenoviral helper genes. We also generated two sets of single-stranded AAV vector constructs carrying an expression cassette containing the EGFP reporter gene flanked by either ITR2 or ITR3. Plasmid transfections of the ITR2- or the ITR3-containing expression cassettes into cultured cells also revealed no difference in the extent of transgene expression. The combination of ITR3 and Rep3 resulted in ~5-fold higher rAAV3 vector titers, as determined by quantitative PCR assays. Next, purified viral stocks of AAV3 vectors were generated and designated as Rep2-ITR2 and Rep3-ITR3, respectively. Interestingly, the transduction efficiency of Rep3-ITR3 AAV3 vectors was ~4-fold higher than that of Rep2-ITR2 AAV3 vectors in a human hepatocellular carcinoma cell line, Huh7, under identical conditions. Additional studies are currently underway to evaluate the efficacy of the Rep2-ITR2 and Rep3-ITR3 vectors in a murine xenograft model in vivo. In summary, our studies document that the combined use of the AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids lead to the production of recombinant AAV3 vectors with higher titers and with higher transduction efficiency, and suggest that the use of homologous ITRs, Rep proteins, and capsids are similarly likely to be applicable to other AAV serotypes vectors for their optimal use in human gene therapy.