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

We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to 1/2 binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (1) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (2) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (3) 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; (4) 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 (5) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE 1/2 binding site, and insertion of 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 AAV vectors, which are serotype, transgene, or promoter agnostic, should be useful in human gene therapy. [Display omitted] Srivastava and colleagues report that ss vectors are largely transcriptionally active since there is no cellular RNA polymerase that can transcribe a ssDNA genome. GenY ssAAV vectors, described here, overcome this major limitation.