Objective: Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease\ngene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single\ninverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here\nwe aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems.\nMethods: Oversized (>8 kb) transgene constructs containing ABCA4 coding sequence were packaged\nas truncated fragments <5 kb in size into various AAV serotypes. In vitro transductions with these fAAV vector\npreparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and\nwestern blot techniques.\nResults: Transductions with fAAV vector preparations yielded ABCA4 mRNA, but did not generate detectable\nlevels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with\nadditional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming\nstable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defined complementary\nregion generated only full length mRNA transcripts plus detectable ABCA4 protein.\nConclusion: Despite previous success shown with the fAAV approach, the lack of repeatability and identification\nof stable hybrid transcripts capable of protein production suggests there is more refinement required before\nconsidering this approach in a clinical setting.
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