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Frank M. Dyka, Shannon E. Boye, Sanford L. Boye, Vince A. Chiodo, William W. Hauswirth; Dual AAV vector system for Usher syndrome (USH1B) gene therapy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1906.
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Usher syndrome type 1B is a severe autosomal recessive deaf-blindness disorder caused by mutations in MYOSINVIIa (MYO7A). MYO7A is expressed in photoreceptors and retinal pigment epithelium (RPE). Due to the relatively large size of the coding gene (6.5 kb), lentiviral vectors have been used to deliver MYO7A transgene to a mouse model of USH1B. However, lentivirus does not efficiently transduce photoreceptors. Adeno-associated viral (AAV) vectors are capable of transducing both photoreceptors and RPE, however AAV has a relatively small packaging capacity (~5kb), thus standard AAV is unsuitable for gene replacement therapy for USH1B. We have previously shown that full length MYO7A expression is achieved using fragmented/"heterogeneous" AAV vector system. However, due to the inability to characterize discrete genetic payloads contained within, this system is unlikely to gain FDA approval. The purpose of this study is to develop a dual AAV vector system with defined genetic components that is capable of delivering full length MYO7A to cells in vitro and in vivo.
Human MYO7A was cloned in AAV vector pairs, where one vector contains a hybrid CMV/chicken βactin promoter and the 5’ portion of MYO7A cDNA sequence and a second vector contains the 3’ portion followed by a polyA signal. Three vector pairs were constructed, where one shares an overlapping sequence of 1350 bp of coding sequence. The other pairs utilize either natural splice sites and intron of MYO7A, or splice donor and acceptor sites of alkaline phosphatase. All vectors were packaged in AAV2 and titer matched for infection of HEK293 cells (10,000 MOI each). Cells were collected 3 days post infection for analysis on immunoblot.
Co-infection with dual MYO7A AAV vectors expressed full length MYO7A in vitro with equal or higher efficiency than that of the "heterogeneous" vector. Of the dual vector systems tested, the overlapping system was the most efficient and mediated expression of only full length MYO7A.
The results show that MYO7A can be expressed using dual AAV vector systems. The system containing overlapping portions of MYO7A was both highly efficient and specific. Because AAV has emerged as the most preferred clinical vector, and because it efficiently transduces both photoreceptors and RPE, our results suggest that a dual AAV vector system may be an option for the treatment of USH1B.
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