1. Inclusion of a degron reduces levelsof undesired inteins after AAV-mediated proteintrans-splicing in the retina
- Author
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Mariangela Lupo, Ivana Trapani, Enrico Maria Surace, Alberto Auricchio, Carlo Gesualdo, Carolina Iodice, Miriam Centrulo, Renato Minopoli, Patrizia Tornabene, Francesca Simonelli, Elena Marrocco, Tornabene, P., Trapani, I., Centrulo, M., Marrocco, E., Minopoli, R., Lupo, M., Iodice, C., Gesualdo, C., Simonelli, F., Surace, E. M., and Auricchio, A.
- Subjects
Genetic enhancement ,viruses ,split-inteins ,QH426-470 ,03 medical and health sciences ,0302 clinical medicine ,Dihydrofolate reductase ,intein degradation ,medicine ,Genetics ,Vector (molecular biology) ,Molecular Biology ,Gene ,030304 developmental biology ,0303 health sciences ,biology ,QH573-671 ,AAV ,Stargardt disease (STGD1) ,medicine.disease ,gene therapy ,Cell biology ,Stargardt disease ,ecDHFR ,inherited retinal disease ,RNA splicing ,030221 ophthalmology & optometry ,biology.protein ,Molecular Medicine ,Original Article ,split-intein ,protein trans-splicing ,Degron ,Intein ,Cytology - Abstract
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., Graphical abstract, Intein-mediated protein trans-splicing (PTS) expands AAV gene transfer capacity. However, non-mammalian inteins persist as trans-splicing by-products. Here, we show that ecDHFR selectively degrades inteins after PTS and that AAV intein vectors armed with this degron are both safe and effective in the retina of a mouse model of genetic blindness.
- Published
- 2021