Enhanced transgene expression from single-stranded AAV vectors in human cells in vitro and in murine hepatocytes in vivo

Abstract

We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to ½ binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (i) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (ii) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (iii) human osteosarcoma cells, U2OS, which lack expression of GR, are poorly transduced by AAV2 vectors, but stable transfection with a GR expression plasmid restores vector-mediated transgene expression; (iv) replacement of the distal 10 nucleotides in the D-sequence of the AAV2 ITR with a full-length GRE consensus sequence significantly enhances transgene expression in human cells in vitro and in murine hepatocytes in vivo, and (v) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE ½ binding site, and insertion of with a full-length GRE consensus sequence in the AAV6-ITR also significantly enhances transgene expression from AAV6 vectors, both in vitro and in vivo. These novel vectors, termed generation Y (GenY) AAV vectors, which are serotype-, transgene-, or promoter-agnostic, should be useful in human gene therapy.