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Frank Dyka, Sanford Boye, Vince Chiodo, Shannon Boye, William Hauswirth; Dual Adeno-associated virus vectors for large cDNA gene replacement therapy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2739. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Adeno-associated virus (AAV) is emerging as the preferred vector to mediate gene replacement therapy due to its low immunogenicity, ability to efficiently transduce a wide variety of cell types and demonstrated efficacy in clinical trials. One of its major drawbacks is a relatively small packaging capacity, ~4.8 kb. Retinal disorders such as recessive Stargardt disease and Usher syndrome 1B are caused by mutations in large genes (ABCA4, MYO7A respectively) that exceed this limit. Alternatively the use of Lentivirus or Adenovirus is problematic due to low transduction efficiency of mature photoreceptors or immunogenicity. After viral entry into the host cell nucleus, dual AAV vectors (cDNA split between two defined vectors) or fragmented (packaging of randomly truncated genomes) AAV vectors recombine to reconstitute full length cDNA. Capitalizing on this we sought to develop defined dual AAV vector systems capable of generating full length cDNAs of large genes in vitro and in vivo.
The coding sequences of MYO7A and ABCA4 were cloned in AAV vector pairs, where one vector contains a CMV/chicken βactin (CBA) promoter and the 5’ portion of the cDNA sequence and a second vector contains the 3’ portion followed by a polyA signal. One vector pair shares a central cDNA sequence of 1.3 kb (simple overlap vector). Another pair utilizes splice donor and acceptor sites (trans-splicing vector) and the third pair utilizes these splice sites in combination with an efficiently recombinogenic overlapping sequence (hybrid vector). All vectors were packaged in AAV2 for transduction of HEK293 cells (10,000 MOI) or retina after subretinal injection into null ABCA4 or MYO7A mice. Cells were collected at 3 days post infection and retina at 6 weeks post infection for analysis.
Co-infection of HEK293 cells with simple overlap, trans-splicing or hybrid AAV vector pairs expressed full length protein in vitro, all with equal or higher efficiency than that of the fragmented vector. Of the three dual vector systems, the hybrid system was the most efficient at expressing full length protein. In-vivo analysis is on-going.
Large cDNAs exceeding the size limitations of conventional AAV can be expressed with high efficiency and specificity using optimally designed dual AAV vector systems. Our results suggest that a dual AAV vector system may be a viable option for the treatment of large gene disorders of the retina.
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