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Livia S Carvalho, Heikki T Turunen, Maria Victoria Luna Velez, Jean Bennett, Luk H Vandenberghe; Comparison of dual AAV vector approaches for large gene delivery to the retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6002.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal gene therapy has come a long way in the last ten years and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for LCA have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV) as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.7kb. Previous studies have demonstrated that homologous recombination and/or ITR concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help package larger constructs. Therefore the main aim of this study was to investigate and compare the use of different AAV dual-vector approaches to deliver large genes to the subretinal space.
We generated an oversized multicistronic AAV construct encoding four reporter genes in a single expression cassette separated by self-cleaving 2A peptides. This was then divided into two separate constructs: left and right, with one of the reporter genes split in half between the two. Three different approaches were tested to evaluate the reconstitution efficiency of the active transcript both in vitro and in vivo in the wild-type mouse retina. Correct reconstitution of the full transcript was assessed by β-galactosidase activity in vitro and by expression of fluorescence reporter genes in the retina.
The three strategies investigated consisted of sequence overlap (OL), trans-splicing (TS) with splice donor and acceptor site and a hybrid (HB) of both. In vitro results showed that all three approaches showed reconstitution levels measured by β-galactosidase activity of 7.7%, 4.3% and 3.4% respectively for the HB, TS and OL strategies. However, analysis of murine retina injected subretinally with the different DUO strategies showed higher reconstitution from TS which was twice as efficient as HB and OL.
This study demonstrates that a dual AAV vector approach is capable of correct transgene reconstitution, although a TS strategy seems to be preferred in vivo compared to a HB approach in vitro. Low efficiency rates and in vivo and in vitro differences however indicate that further study is needed to better understand the reconstitution mechanisms behind a dual AAV strategy.
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