Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina

Abstract

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein–mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

An expanded library of orthogonal split inteins enables modular multi-peptide assemblies

“…DNA constructions were performed according to the principles of Gibson assembly 55 or using the BioBrick RFC [10] standard 56 , following standard molecular biology techniques. For the BioBrick RFC [10] standard FastDigest EcoRI, BcuI (SpeI), PstI, and XbaI enzymes were used (FD0274, FD1253, FD0614, and FD0684, respectively; Thermo Fisher). DNA fragments were generated by polymerase chain reaction (PCR) amplifications using primers with ≥18 bp overlaps.…”

“…The ability of inteins to seamlessly ligate two peptides or to selectively release the N-or C-extein portion has been extensively exploited for protein modification and purification, to produce intein-based biosensor and reporter systems, and even in gene therapy [6][7][8][9][10][11][12][13] .…”

Modelling and rescue of RP2 Retinitis Pigmentosa using iPSC Derived Retinal Organoids

“…The use of AAV in human ROs is relatively limited at the moment, but studies have reported varying efficiency of transduction, which could be attributed to age of treatment, time of harvesting, vector tropisms, viral titre, the promotor used and sensitivity of detection of the transgene, e.g. antibody versus intrinsic fluorescence of a reporter (Gonzalez-Cordero et al, 2018, Tornabene et al, 2019, Quinn et al, 2019, Khabou et al, 2018. Here we demonstrate highly efficient transduction using an AAV2/5 vector with RP2 under the control of a CAG promoter.…”

“…AAVs are able to transduce post-mitotic rods and cones in mice following sub-retinal or intravitreal injection (Sarra et al, 2002) and in iPSC derived rod and cone photoreceptors in ROs with varying efficiency (Gonzalez-Cordero et al, 2018, Tornabene et al, 2019. In order to assess the ability of AAVs to deliver RP2 to deficient photoreceptors, RP2 KO ROs were transduced with AAV2/5.CAGp.RP2 (Fig.…”

Can Adeno-Associated Viral Vectors Deliver Effectively Large Genes?

“…Delivery of AAVintein vectors encoding for the ABCA4 protein in a mouse model of Stargardt disease resulted in significant amelioration of the phenotype. 99 Importantly, in a side-by-side comparison between the AAV-intein and dual AAV hybrid platforms, we found that the levels of AAV inteinmediated large protein reconstitution largely exceeded those achieved via AAV genome recombination by dual AAV vectors. 99 In addition, in the large pig retina we have found levels of AAV-intein-mediated protein reconstitution comparable to those achieved with a single AAV vector.…”