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

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.

Liver gene therapy with intein‐mediated F8 trans ‐splicing corrects mouse haemophilia A

“…The excised inteins bioproduct could raise immunological concerns as it is non‐mammalian; however, no apparent toxicity has been observed in previous studies (Chen et al , 2007 ; Zhu et al , 2010 , 2011 , 2013 ; Tornabene et al , 2019 ) nor in ours, though our study was not designed to assess potential toxicity. Independently, we have recently incorporated a degron in the AAV‐intein vectors that results in selective intein degradation following PTS, therefore without significantly impacting on levels of full‐length protein (Tornabene et al , 2021 ). This could be incorporated in the future in the AAV‐N6 intein vectors, if necessary.…”

Utility of the DHFR-based destabilizing domain across mouse models of retinal degeneration and aging

“…Moreover, except for the aged mice, we evaluated mice at a single time point during the retinal degeneration, which does not account for additional physiological changes that may occur as the disease starts to progress. Future longitudinal studies could shine light onto the long-term stability and biological tolerance for the DHFR DD, although a recent study analyzed mice subretinally injected with an AAV encoding for a mini DHFR degron without any apparent detriment out to 1 year of age ( Tornabene et al., 2021 ). In addition, a more thorough examination of whether particular retinal cell types are recalcitrant to DHFR DD regulatability or whether signal from readily transduced/numerous cells masks the signals from less abundant/less transduced cell types are important aspects to consider.…”

Inteins as Drug Targets and Therapeutic Tools

“…As expected, lipofuscin was observed to accumulate in the retinal pigmented epithelium of the vehicle-treated Abca4 −/− mice. In contrast, there was a significant reduction in lipofuscin, in addition to improvement in other phenotypes for the mice treated with ABCA4-split intein after 3 months ( Tornabene et al, 2021 ). Similarly, studies with the retina of pig and human retinal organoids also showed improvement for the ABCA4-split-intein group compared with the vehicle controls ( Tornabene et al, 2021 ).…”

“…ABCA4 is involved in the clearance of photoisomerized all-trans-retinal from the photoreceptor disk lumen. Mutated ABCA4 results in a buildup of lipofuscin pigments in the retinal pigment epithelial cells, causing vision loss in patients with Stargardt disease ( Tornabene et al, 2021 ). In order to repair the ABCA4 mutation, the ABCA4 gene is split into two; and each half is linked to split inteins which are separated, loaded onto AVV vectors, and delivered to one-month-old Abca4 −/− mice subretinally.…”

“…The specific signals which turn the protein susceptible to ubiquitin-mediated proteasomal degradation are called degrons ( Varshavsky, 1991 ). Inclusion of degron E. coli dihydrofolate reductase (ecDHFR) in the N-intein results in selective degradation of excised inteins from the AVV vector that is used for delivery of the ABCA4 gene for retinal therapy ( Tornabene et al, 2021 ). The degradation ability of ecDHFR is inhibited by a small stabilizing ligand, trimethoprim.…”