AAV-mediated protein trans-splicing in the retina

Abstract

Purpose : Retinal gene therapy with AAV vectors is safe and effective, yet it is limited by AAV cargo capacity of about 5 kb. To overcome this limitation we explored the use of intein-mediated protein trans-splicing to reconstitute large proteins in the retina. Inteins work as independent peptides fused to the C- and N- termini of two host proteins (i.e. the two halves of a large protein) and mediate their association in a multistep autocatalytic process.

Methods : To test protein trans-splicing in the retina, we generated two AAV vectors separately encoding each of the two halves of either the reporter EGFP protein or large therapeutic proteins flanked by split-inteins. These include ABCA4 and CEP290, respectively defective in Stargardt disease (STGD1) and Leber congenital amaurosis 10 (LCA10), two severe and common inherited blinding diseases. We identified in each protein optimal splitting points for the generation of AAV-intein constructs which take into account both amino acid residue requirements for trans-splicing to occur, as well as the preservation of the native protein domains.

Results : Upon co-administration of both AAV split-intein vectors, full-length proteins were reconstituted in the mouse and pig retina as well as in human retinal organoids derived from induced pluripotent stem cells. Importantly, the levels of large protein reconstitution achieved reduced lipofuscin accumulation and retinal degeneration in the mouse models of STGD1 and LCA10, respectively.

Conclusions : Our data support the use of intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.