title: A fragmented adeno-associated viral dual vector strategy for treatment of diseases caused by mutations in large genes leads to expression of hybrid transcripts.
abstract: OBJECTIVE
  Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems.
  
  
  METHODS
  Oversized (>8kb) transgene constructs containingABCA4coding sequence were packaged as truncated fragments <5kb in size into various AAV serotypes.In vitrotransductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques.
  
  
  RESULTS
  Transductions with fAAV vector preparations yieldedABCA4mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full lengthABCA4CDS with additional hybridABCA4variants, indicating truncated transgenes without regions of overlap were joining and forming stable hybrid transgenes. In contrast, anABCA4overlapping dual vector system (OV) with a defined complementary region generated only full length mRNA transcripts plus detectable ABCA4 protein.
  
  
  CONCLUSIONS
  Despite previous success shown with the fAAV approach, the lack of repeatability and identification of stable hybrid transcripts capable of protein production suggests there is more refinement required before considering this approach in a clinical setting.
authors.affiliation: Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Michelle E
authors.initials: ME
authors.lastname: McClements
authors.affiliation: Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK; Oxford Eye Hospital, The John Radcliffe Hospital, Oxford, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Charbel Issa
authors.affiliation: Laboratoire de Thérapie Génique UMR1089, Structure Federative de Recherche - IRS1, Quai Moncousu, BP 70721, 44007 Nantes cedex.
authors.forename: Véronique
authors.initials: V
authors.lastname: Blouin
authors.affiliation: Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK; Oxford Eye Hospital, The John Radcliffe Hospital, Oxford, UK.
authors.forename: Robert E
authors.initials: RE
authors.lastname: MacLaren