310. Development of Generation X Recombinant AAV Vectors for Human Gene Therapy
Authors: Chen Ling, Yuan Lu, Yuanhui Zhang, Baozheng Li, Wenqin Ma, George V. Aslanidi, Changquan Ling, Arun Srivastava
Journal: Molecular Therapy (2015)

We have previously reported the development of capsid-modified next generation (NextGen) AAV serotype vectors that transduce cells and tissues more efficiently at reduced vector doses (Proc. Natl. Acad. Sci., USA, 105: 7827-7832, 2008). More recently, we have also described the development of genome-modified generation X (GenX) AAV vectors that also transduce cells and tissues more efficiently (J. Virol, 89: 952-961, 2015). The recombinant AAV genome contains inverted terminal repeats (ITRs) of 145 nucleotides at both ends. In these studies, a 20-nucleotide sequence, termed the D-sequence, was replaced with a substitute sequence, which led to enhanced transgene expression in human cell lines in vitro an in murine hepatocytes in vivo (J. Virol., 89: 952-961, 2015). In our present studies, we observed that a sequence, GGTTCCT, at the end of the D-sequence, shares partial homology to the consensus glucocorticoid receptor-binding element (GRE) site, 5'-GGTACANNNTGTT/CCT-3'. The TGTTCT half-site is an essential core element, which has been reported to be sufficient to relay glucocorticoid signaling. In electrophoretic mobility-shift assays (EMSAs), we documented that purified GR protein could specifically bind to double-stranded D-sequence oligonucleotides, suggesting that the D-sequence potentially functions as a ½ GRE site. Based on these results, we hypothesized that replacement of the D-sequence with a full GRE binding-site in the ITR might further increase transgene expression from these GenX AAV genomes. To this end, recombinant AAV vectors were generated in which the D-sequence was replaced with a fully functional GRE site. Insertion of a full GRE binding-site in the ITR significantly increased the transgene expression from these GenX AAV genomes following encapsidation in the wild-type (WT) AAV2 capsid vectors in human cell lines in vitro, and the extent of the transgene expression was further increased by dexamethasone-treatment. When 1×1010 vgs of recombinant AAV2 vectors containing the Gaussia luciferase (Gluc) reporter gene in the unmodified AAV genome, or those containing the GRE sequence, were administered via tail-vein into C57BL6/J mice, the transduction efficiency of the AAV-GRE vectors was ~8-fold higher in murine hepatocytes in vivo up to 9-weeks post-vector administration. More interestingly, when AAV-GRE genomes containing the enhanced green fluorescence protein (EGFP) reporter gene were encapsidated in the optimal NextGen AAV capsid-modified quadruple-mutant (Y444F+Y500F+Y730F+T491V) AAV2 vectors, the transduction efficiency of these vectors was further increased by ~8-fold in murine hepatocytes in vivo at a dose as low as 5×108 vgs/mouse. Taken together, the availability of these novel GenX AAV vectors containing the D-sequence substitution, and the fully functional GRE site insertion, to achieve high-efficiency transgene expression, has implications in the use of these vectors in human gene therapy.