Author(s): Ivana Trapani 1 2 , Patrizia Tornabene 1 2 , Alberto Auricchio 1 3
(1) Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy
(2) Medical Genetics, Department of Translational Medicine, Federico II University, Naples, Italy
(3) Department of Advanced Biomedicine, Federico II University, Naples, Italy
Retinal gene therapy is witnessing an unprecedented clinical development, one of the most significant in the field, boosted by the recent Luxturna market authorization  and the favorable characteristics of the eye . The majority of gene therapy products under development for retinal diseases are based on adeno-associated viral vectors (AAV) , given their unmatched ability to efficiently target retinal neurons upon intravitreal or subretinal administration . However, one of the main drawbacks of AAV is their ~5 kb DNA packaging capacity , which limits their applications, in particular considering that effective gene expression needs regulatory elements in addition to a therapeutic coding sequence (CDS).
In order to overcome this limitation, a large CDS may be split in separate AAV vectors, which upon co-transduction of the same target cell reconstitute the full-length transgene expression cassette or protein [5-8]. In one strategy, a large expression cassette is split in two AAV genomes (dual AAV, Fig. 1) each containing one half of the expression cassette: the 5' vector contains both the promoter and the 5' half CDS while the 3' vector contains the 3' half CDS with the polyadenylation signal. The recombination of the two vectors and the reconstitution of the full-length expression cassette occurs through either: (i) sequences of the transgene which are repeated both at the end of the 5' vector and at the beginning the 3' and trigger homologous recombination (overlapping dual AAV) ; (ii) the AAV inverted terminal repeats (ITR), which inherently undergo intermolecular recombination thus mediating the concatemerization of two different AAV genomes (trans-splicing dual AAV) ; or (iii) exogenous short highly recombinogenic sequences included both at the end of the 5' vector and at the beginning of the 3' which favor tail-to-head concatemerization of the two genomes (hybrid dual AAV) . Both trans-splicing and hybrid dual AAV also carry splicing signals after the 5' half CDS and before the 3' half CDS to splice out the ITR and the exogenous recombinogenic sequences from the full-length transgene transcript once recombination has occurred.
Split AAV-based strategies to restore large gene expression.
Large coding sequences (CDS) can be split across separate AAV vectors (AAV I and AAV II) which in transduced cells reconstitute the full-length protein through either DNA recombination (dual AAV vectors) or protein trans-splicing (AAV intein vectors). pA polyadenylation signal, Int intein.
Various groups have independently reported that dual AAV overlapping, trans-splicing and hybrid vectors efficiently reconstitute large genes in mouse and pig photoreceptors to levels which resulted in a significant improvement in the phenotype of mouse models of common inherited retinal diseases (IRDs) [5-8, 12]....