Research ArticleGene Therapy

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

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Science Translational Medicine  15 May 2019:
Vol. 11, Issue 492, eaav4523
DOI: 10.1126/scitranslmed.aav4523

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Leveraging naturally occurring splicing for gene therapy

Retinal gene therapy using adeno-associated viral (AAV) vectors has been shown to be effective in a number of eye diseases. However, the limited capacity of AAV hampers the use of gene therapy for delivering large genes. Now, Tornabene et al. leveraged the properties of genetic elements called split inteins to deliver large genes using multiple vectors. In unicellular organisms, split inteins mediate the fusion of two separate peptides. Here, the authors were able to reconstitute large full-length proteins using split inteins in vitro in pig retina and human organoids and in vivo in two mouse models of inherited retinal diseases with high efficiency, obtaining therapeutic effects.


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.

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