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Frank M Dyka, Sanford L Boye, William W Hauswirth, Shannon Boye; Characterization of fidelity of dual Adeno-associated virus vectors generated cDNA. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3330.
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© ARVO (1962-2015); The Authors (2016-present)
Adeno-associated virus (AAV) has proven success in gene transfer for clinical applications due to its ability to transduce a wide variety of cell types and its low immunogenicity. A drawback, however, is its small packaging capacity of <5 kb. Mutations in a gene that exceeds this limit, MYO7A for example, are associated with the most common and severe form of Usher syndrome (USH1b). To express large genes, dual AAV vector strategies, in which the cDNA is split between two vectors has emerged as a valuable tool. We have previously shown that MYO7A is efficiently expressed from hybrid, trans-splicing and simple overlap dual vector platforms in vitro and in vivo. However, mechanisms by which these dual vectors produce full length MYO7A (homologous recombination and/or mRNA splicing) have the potential to introduce mutations. Missense mutation and truncation alleles for MYO7A cause a more severe phenotype than mutations resulting in null alleles. Therefore, clinical translatability of dual AAV vectors may depend on sequence fidelity of the recombined full length transgene. Here, we evaluate the sequence fidelity of MYO7A transcripts arising from different dual AAV vector platforms.
Human MYO7A was cloned in AAV vector pairs, where one vector contained a promoter and the 5’ portion of the cDNA sequence and a second vector contained the 3’ portion followed by a polyA signal. “Simple overlap” vectors share a central 1.3 kb sequence of MYO7A. “Hybrid” and “trans-splicing” vectors contain splice donor and acceptor sites with and without an additional sequence for recombination, respectively. All vectors were packaged separately for transduction in HEK293 cells. Cells were collected 3 days post infection for analysis of protein and mRNA expression. mRNA was reverse transcribed and analyzed by restriction endonuclease treatment and sequencing.
Infection of HEK293 cells with the simple overlap, trans-splicing or hybrid AAV dual vector platforms expressed full length protein in vitro. Endonuclease treatment and sequencing of the critical areas of recombination and/or the splice region showed fidelity of the MYO7A mRNA with no missense or frame shift mutations detectable.
Large cDNAs exceeding the size limitations of AAV can be expressed with high efficiency and specificity using dual AAV vectors. Our results show that the sequence of MYO7A mRNA produced from dual AAV vectors is intact.
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