Development Of Universal, Strong Mini-Promoters For Recombinant Adeno-Associated Viral (Raav) Vectors

rAAVs have emerged as an efficient gene delivery tool. Discovery of various natural serotypes and recent development of recombinant capsids significantly advanced the transduction efficiency of rAAVs in a variety of cells and tissues. On the other hand, much less effort has been made for maximizing expression of the rAAV cargo DNA, since current AAV vectors mainly rely on well-established promoters for gene expression. Among those, CMV and CAG promoters belong to the most frequently used strong promoters providing universal activity. The capacity of DNA packaging in rAAV capsids is limited (4.7 kb). Hence, the large size of the existing strong promoters is a drawback in delivering genes and gene editing tools of large sizes, reaching the limits of the viral packaging capacity. To improve AAV as a gene therapy tool, discovery of small but strong promoters is a crucial step. Here we report two new strong mini promoters, called INS84 and GCG110, with universal activity in rAAV expression vectors. These promoters are only 84 and 135 base pairs in size, respectively. They showed strong expression of a reporter transgene from rAAV in human and mouse cells and tissues, including human hepatocytes in primary cultures, humanized mice in vivo and human pancreatic islet cells. Expression levels in these tissues were comparable to those obtained with the much larger CAG promoter. Until now, viral vectors utilized (or ‘borrowed’) promoters that are characterized in the context of plasmid expression vectors or germ-line transgenes. Our strong mini-promoters for rAAV expression suggest a new direction for developing promoters for viral vectors, specifically that the large size of promoters required for expression in the context of plasmid vectors is often not necessary for strong expression in an rAAV vector.