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

Yuan Lu; Chen Ling; Jakob Shoti; Hua Yang; Aneesha Nath; Geoffrey D Keeler; Keyun Qing; Arun Srivastava

doi:10.1016/j.omtn.2024.102196

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

Mol Ther Nucleic Acids. 2024 Apr 23;35(2):102196. doi: 10.1016/j.omtn.2024.102196. eCollection 2024 Jun 11.

Authors

Yuan Lu¹, Chen Ling², Jakob Shoti^{3

4}, Hua Yang⁵, Aneesha Nath⁶, Geoffrey D Keeler³, Keyun Qing³, Arun Srivastava^{3

4

7}

Affiliations

¹ Full Circle Therapeutics, Shanghai, China.
² Department of Genetics, School of Life Sciences, Fudan University, Shanghai, China.
³ Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA.
⁴ Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.
⁵ Department of Radiology, The Third Xiangya Hospital, Central South University, Changsha, China.
⁶ Department of Pharmacotherapy & Translational Research, University of Florida College of Pharmacy, Gainesville, FL, USA.
⁷ Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA.

Abstract

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.

Keywords: AAV vectors; MT: Delivery Strategies; gene therapy; gene transfer; glucocorticoid receptor-binding element; transgene expression.