AAV split intein

Adeno-associated viral (AAV) vectors are thus far considered the leading platform to introduce genes in retinal cells for gene therapy purposes. However, their broad application is hampered by a packaging capacity <5 kb, which is insufficient for the delivery of large genes causative of common retinal diseases, such as EYS and USH2A. To overcome this limitation, we will explore protein trans-splicing mediated by split-inteins. Split-inteins are elements independently expressed at the extremities of separate proteins, which are capable of recognize each other and, through the process of protein trans-splicing, mediate ligation of the flanking proteins and their own removal. We have already shown that delivery of multiple AAV vectors each encoding one of the fragments of a large protein flanked by split-inteins results in full-length protein reconstitution in the retina of animal models and in three-dimensional cellular systems resembling the human retina. Here, we thus propose to further explore this platform for reconstitution of the large EYS and Usherin proteins. To this aim, we will generate and test various sets of AAV-intein vectors, in vitro, in order to select the most efficient for EYS and Usherin proteins reconstitution. Next, we will define the efficiency of the platforms both in the retina of animal models and in three-dimensional cellular systems.