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SJ Reich, AM Maguire, A Auricchio, M Hildinger, JM Wilson, J Bennett; Trans-Splicing AAV Vector System Expands the Packaging Capacity Of AAV Gene Therapy Vectors for Delivery of Large Transgenes to the Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4589.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Adeno-Associated Virus (AAV) based recombinant vectors can safely transduce different retinal cell types resulting in efficient gene expression. A major shortcoming of this vector is its small packaging capacity that limits the delivery of large transgene cassettes. A trans-splicing approach has been developed, which reconstitutes gene expression from two independent AAV vectors, in order to overcome the vector's packaging restrictions. Our aim was to determine whether this trans-splicing AAV system would work in the context of the retina. Methods: AAV2.1lacZ-SD contains the upstream half of the lacZ cDNA followed by a splice donor site under the control of the cytomegalovirus (CMV) promoter. AAV2.1lacZ-SA contains the downstream half of the lacZ cDNA initiated by a splice acceptor site. Both vectors were mixed at a 1:1 ratio and delivered unilaterally to eyes of CD1 mice by subretinal or intravitreal injection. The contralateral eyes received AAV carrying the gene encoding the intact lacZ gene as control. AAV2.1lacZ-SD and AAV2.1lacZ-SA were also delivered individually to eyes of additional CD1 mice to show there is no gene expression when the vectors are used alone. At 2-6 weeks post-injection, the mice were sacrificed and the effects of the treatments were evaluated by histochemical assay. Results: By bright field microscopy, lacZ expression was evident as early as 2 weeks and the level of expression increased with time up to 4 weeks. Expression persisted through the latest timepoint (6 weeks). All retinal cells known to be targeted by AAV2 were positive for transgene expression, including retinal pigment epithelium cells, photoreceptors and ganglion cells. Eyes from mice treated with either AAV2.1lacZ-SD or AAV2.1lacZ-SA alone showed no lacZ expression. Eyes from mice treated with the AAV.lacZ control vector were similar except that lacZ expression was significantly stronger and more widespread than in experimentals at every timepoint. Conclusion: The trans-splicing AAV vector system can transduce cells of the retina when delivered by sub-retinal injection. This supports the potential utility of trans-splicing AAV as a vector for the treatment of diseases of the retina that require delivery of genes that exceed the packaging limits of conventional AAV vectors.
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