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Frank M Dyka, Vince A. Chiodo, William W Hauswirth; Serotype dependent Adeno-Associated Virus Hybrid Vector mediated gene therapy for Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2464.
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© ARVO (1962-2015); The Authors (2016-present)
Adeno-associated virus (AAV) hybrid vectors have been shown to overcome the size limitations 4.8kb of a single AAV capsid by splitting the coding sequence (cds) of an oversized gene between two vectors. The retina specific ATP binding cassette transporter A4 (ABCA4) which is necessary for the clearance of all-trans-retinal from photoreceptor cells has a cds of 6.8kb thus exceeding this limit. Loss of ABCA4 function results to accumulation of toxic bisretinoids and ultimately leads to the Stargardt phenotype of increased autofluorescence and progressive RPE and photoreceptor cell loss.Here we compare the performance of two highly efficient AAV capsid serotypes for ABCA4 gene replacement therapy in an ABCA4 knock out mouse model of Stargardt disease and test the effect of hybrid vector treatment in older mice.
The human ABCA4 cds was cloned into an AAV hybrid vector pair. The 5’ vector carries a ubiquitous promoter, the 5’ part of the ABCA4 cds, a splice donor site and a short highly recombinogenic sequence. The 3’ vector contains the same recombinogenic sequence, a splice acceptor site, the 3’ part of the ABCA4 cds and a polyadenylation signal. The constructs were packaged in the improved AAV8(Y733F) and AAV2/3YF (Y444F,Y500F,Y730F) capsids. Protein expression in treated and untreated ABCA4 knock out mice was determined by western blot and correct localization by IHC. Sets of 3 month old ABCA4 knock out mice were subretinally injected with ABCA4 hybrid vector pairs in one eye. The effect of AAV hybrid vector treatment was analyzed 1 and 3 months post injection by measuring the lipofuscin autofluorescence with a confocal scanning laser ophthalmoscope and the integrity of retinal layering by optical coherence tomography. Statistical significance was determined with Student’s t-test with p>0.05 considered significant.
ABCA4 hybrid vectors packaged in different serotypes expressed full length ABCA4 protein and the protein localized correctly to the outer segments of photoreceptors. However the number of transduced cells was significantly higher when using the AAV8(Y733F) vector capsid vs. the AAV2/3YF vector capsid.
cDNAs exceeding the payload capacity of AAV can be delivered by AAV hybrid vectors with high efficiency and specificity to photoreceptor cells utilizing the AAV8(Y733F) serotype. The AAV2/3YF however did not produce significant amounts of ABCA4 protein.
This is a 2020 ARVO Annual Meeting abstract.
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