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Frank M Dyka, William W Hauswirth; Dual Adeno-Associated Virus Vector Treatment of Autosomal Recessive Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4533. doi: https://doi.org/.
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The ATP binding cassette transporter A4 (ABCA4) is involved in the clearance of all-trans-retinal from photoreceptor outer segments. It functions as a flippase moving N-retinylidene-PE from the inner to outer leaflet of the lipid bilayer. Loss of function causes the accumulation of the toxic side product N-retinylidene-N-retinylethanolamine (A2E) in the retinal pigment epithelium and ultimately leads to the Stargardt disease phenotype. This distinct genotype-phenotype relationship makes ABCA4-Stargardt disease a viable target for gene replacement therapy. Adeno-associated virus vectors have shown great potential for gene transfer to a variety of post-mitotic cell types with high efficiency. The ABCA4 coding sequence (cds) of 6.8 kb exceeds the AAV payload limit of 4.8kb by far, making it impossible to deliver the transgene in a single AAV capsid. AAV dual vectors can overcome this size restriction. In these vectors the cds is split between two vectors and packaged in separate capsids. After co-infection the two genomes come together through homologous recombination or non-homologous end joining to reconstitute the full length coding sequence. Here we present data on the effect of AAV dual vector mediated ABCA4 gene therapy in an ABCA4 knock out mouse.
The human ABCA4 cds was cloned into an AAV dual vector pair. The 5’ vector contained the promoter and the 5’ portion of the ABCA4 cds. The 3’ vector contained the 3’ portion of the ABCA4 cds and a polyadenylation signal. Both vectors share the same highly recombinogenic element. Sets of ABCA4 knock out mice were injected subretinally with AAV dual vectors or the 5’ vector alone. Expression of protein was detected by western blot and the treatment effect measured up to 5 months post injection by detection of lipofuscin/A2E autofluorescence with a confocal scanning laser ophthalmoscope.
The AAV hybrid dual vectors expressed full length ABCA4 in vitro and in vivo. Fundus autofluorescence measurements showed a significant reduction in lipofuscin accumulation in dual vector treated versus 5’ vector only treated animals.
AAV hybrid dual vectors are capable of delivering cDNAs exceeding the payload capacity of AAV with high efficiency and specificity. Our results indicate that AAV hybrid vector delivered ABCA4 has a significant effect on lipofuscin/A2E accumulation in the ABCA4 knock out mouse.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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