A Fragmented Adeno-Associated Viral Dual Vector Strategy for Treatment of Diseases Caused by Mutations in Large Genes Leads to Expression of Hybrid TranscriptsMichelle E McClements1*, Peter Charbel Issa1,2, Véronique Blouin3and Robert E MacLaren1,2
- *Corresponding Author:
- Robert E MacLaren
Nuffield Laboratory of Ophthalmology
Nuffield Department of Clinical Neurosciences
University of Oxford, Oxford, UK
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E-mail: [email protected]
Received date: October 18, 2016; Accepted date: November 07, 2016; Published date: November 14, 2016
Citation: McClements ME, Issa PC, Blouin V, MacLaren RE (2016) A Fragmented Adeno-Associated Viral Dual Vector Strategy for Treatment of Diseases Caused by Mutations in Large Genes Leads to Expression of Hybrid Transcripts. J Genet Syndr Gene Ther 7:311. doi: 10.4172/2157-7412.1000311
Copyright: © 2016 McClements ME, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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 containing ABCA4 coding sequence were packaged as truncated fragments <5kb in size into various AAV serotypes. In vitro transductions 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 yielded ABCA4 mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full length ABCA4 CDS with additional hybrid ABCA4 variants, indicating truncated transgenes without regions of overlap were joining and forming stable hybrid transgenes. In contrast, an ABCA4 overlapping dual vector system (OV) with a defined complementary region generated only full length mRNA transcripts plus detectable ABCA4 protein. Conclusion 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.