Inclusion of a degron reduces levels of undesired inteins after AAV-mediated protein trans-splicing in the retina

• DOI: 10.1016/j.omtm.2021.10.004
• Publication Date: 2021-10-22T07:25:00Z
• Authors: Patrizia Tornabene; Ivana Trapani; Miriam Centrulo; Elena Marrocco; Renato Minopoli; Mariangela Lupo; Carolina Iodice; Carlo Gesualdo; Francesca Simonelli; Enrico M. Surace; Alberto Auricchio
• Keywords: 0301 basic medicine; QH573-671; split-inteins; AAV; Stargardt disease (STGD1); QH426-470; gene therapy; 03 medical and health sciences; ecDHFR; inherited retinal disease; intein degradation; Genetics; AAV; ecDHFR; gene therapy; inherited retinal disease; intein degradation; protein trans-splicing; split-inteins; Stargardt disease (STGD1); Original Article; protein trans-splicing; Cytology

Split intein-mediated protein trans-splicing expands AAV transfer capacity, thus overcoming the limited AAV cargo. However, non-mammalian inteins persist as trans-splicing by-products, and this could raise safety concerns for AAV intein clinical applications. In this study, we tested the ability of several degrons to selectively decrease levels of inteins after protein trans-splicing and found that a version of E. coli dihydrofolate reductase, which we have shortened to better fit into the AAV vector, is the most effective. We show that subretinal administration of AAV intein armed with this short degron is both safe and effective in a mouse model of Stargardt disease (STGD1), which is the most common form of inherited macular degeneration in humans. This supports the use of optimized AAV intein for gene therapy of both STGD1 and other conditions that require transfer of large genes.